Ehrlichia ewingii proteins, nucleic acids, and methods of their use

ABSTRACT

The novel omp-1 gene cluster encoding twenty one  Ehrlichia ewingii  (EE) proteins was isolated and sequenced completely. This invention relates to isolated  E. ewingii  (EE) polypeptides, isolated polynucleotides encoding EE polypeptides, probes, primers, isolated antibodies and methods of their production, immunogenic compositions and vaccines, as well as methods of using the EE polypeptides, antibodies, probes, and primers for the purpose of diagnosis, therapy and production of vaccines against  E. ewingii.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/115,490, filed May 5, 2008, now U.S. Pat. No. 8,784,828, which claimspriority to US Provisional Application Ser. No. 60/916,227, filed May 4,2007; and U.S. Provisional Application Ser. No. 61/016,348, filed Dec.21, 2007; the entire contents of which are incorporated herein byreference.

GOVERNMENT RIGHTS

This invention was made, at least in part, with federal funding from theNational Institutes of Health Grant R01A147407. The United StatesGovernment may have certain rights in this invention.

BACKGROUND

Ehrlichia ewingii, a tick-transmitted rickettsia previously known onlyas a canine pathogen, is the most recently recognized human granulocyticehrlichiosis agent. Granulocyte-tropic Ehrlichia was first reported byDr. S. A. Ewing in 1971 in a dog from Arkansas and was thought to be agranulocytic variant of Ehrlichia canis. Granulocyte-tropic Ehrlichiawas recognized as a separate species in 1992, based on 16S rRNA genesequence comparison and named as Ehrlichia ewingii in honor of Dr. S. A.Ewing. Since then, canine infection with E. ewingii has been reported inseveral states in the U.S. and recently from Africa. Clinical signs indogs infected with E. ewingii are fever, lethargy, anorexia, lameness,and polyarthritis, accompanied with mild thromobocytopenia and mildanemia. In 1999, human infection with E. ewingii was documented. Since1996, retrospectively, approximately 10 confirmed cases of humangranulocytic ehrlichiosis caused by E. ewingii infection have beenidentified in Missouri and Oklahoma.

Diagnosis of E. ewingii infections has proven difficult. E. ewingii hasyet to be cultivated, and there is no serologic test available todiagnose E. ewingii infection. Clinical signs of patients infected withE. ewingii, such as fever, headache, myalgia, leukopenia, andthrombocytopenia are similar to those of human monocytic ehrlichiosiscaused by E. chaffeensis and human granulocytic anaplasmosis caused byA. phagocytophilum. Hence, clinical features alone cannot distinguishthese causative agents. Further complicating the diagnosis ofehrlichiosis infections, E. ewingii and E. chaffeensis also share thesame vector tick species and animal reservoirs. Experimentally, the Lonestar tick (Amblyomma americanum) has been shown to be a competentvector, although bacterial DNA has been detected in other species ofticks. White-tailed deer (Odocoileus virginianus) is considered to be animportant reservoir for E. ewingii and dogs are also possiblereservoirs. Consequently, E. ewingii and E. chaffeensis have similarseasonal and geographic distributions. While bacteria have been seen onblood smears from infected animals and humans, and detected by PCR inthe blood and tick specimens, to date E. ewingii remains uncultivableand a stable laboratory isolate is not available. PCR tests based on theE. ewingii-specific partial sequence of a 16S rRNA gene and a partialp28-19 sequence have been reported (Gusa, A. A., et al. 2001. J. ClinMicrobiol 39:3871-3876). Yet, sensitivities and specificities of E.ewingii PCR tests in clinical specimens are unknown, as there are noother definitive tests with which to compare. The microscopicobservation of morulae in Romanovsky dye-stained peripheral bloodgranulocytes provides definitive proof of ehrlichial infections.Unfortunately, this test cannot be used as a single diagnostic test forE. ewingii infection because it cannot distinguish E. ewingii morulaefrom other granulocyctic agents, such as A. phagocytophilum.Furthermore, negative results from Romanovsky dye-staining cannot ruleout E. ewingii infection, owing to high false-negative rates caused bysample conditions and the low sensitivity of the assay. These setbacksin prior diagnostic testing necessitate an additional test to properlyidentify E. ewingii infection.

SUMMARY

Provided herein is an isolated E. ewingii (EE) polypeptide that includesan amino acid sequence of a mature EE protein or a functional derivativethereof. The mature EE protein is selected from the group consisting of:(1) amino acid 24 to 293 of SEQ ID NO 3 corresponding to a matureOMP-1-1 protein encoded by nucleotide 67203-1484 of SEQ ID NO: 1; (2)amino acid-22 to 272 of SEQ ID NO: 4 corresponding to a mature OMP-1-2protein encoded by nucleotide 2116-2871 of SEQ ID NO: 1; (3) amino acid24 to 284 of SEQ ID NO: 6 corresponding to a mature OMP-1-3 proteinencoded by nucleotide 3610-4395 of SEQ ID NO: 1; (4) amino acid 28 to293 of SEQ ID NO: 7 corresponding to a mature OMP-1-4 protein encoded bynucleotide 4486-5286 of SEQ ID NO: 1; (5) amino acid 24 to 272 of SEQ IDNO: 8 corresponding to a mature OMP-1-5 protein encoded by nucleotide5380-6129 of SEQ ID NO: 1; (6) amino acid 26 to 299 of SEQ ID NO: 9corresponding to a mature OMP-1-6 protein encoded by nucleotide6216-7040 of SEQ ID NO: 1; (7) amino acid 27 to 284 of SEQ ID NO: 10corresponding to a mature OMP-1-7 protein encoded by nucleotide7145-7921 of SEQ ID NO: 1; (8) amino acid 29 to 243 of SEQ ID NO: 11corresponding to a mature OMP-1-8 protein encoded by nucleotide8032-8679 of SEQ ID NO: 1; (9) amino acid 28 to 281 of SEQ ID NO: 12corresponding to a mature OMP-1-9 protein encoded by nucleotide8772-9536 of SEQ ID NO: 1; (10) amino acid 26 to 280 of SEQ ID NO: 13corresponding to a mature OMP-1-10 protein encoded by nucleotide9620-10387 of SEQ ID NO: 1; (11) amino acid 28 to 290 of SEQ ID NO: 14corresponding to a mature OMP-1-11 protein encoded by nucleotide10477-11268 of SEQ ID NO: 1; (12) amino acid 27 to 298 of SEQ ID NO: 15corresponding to a mature OMP-1-12 protein encoded by nucleotide11370-12188 of SEQ ID NO: 1; (13) amino acid 30 to 302 of SEQ ID NO: 16corresponding to a mature OMP-1-13 protein encoded by nucleotide12292-13113 Of SEQ ID NO: 1; (14) amino acid 26 to 285 of SEQ ID NO: 17corresponding to a mature OMP-1-14 protein encoded by nucleotide14530-15312 of SEQ ID NO: 1; (15) amino acid 26 to 278 of SEQ ID NO: 18corresponding to a mature OMP-1-15 protein encoded by nucleotide15689-16450 of SEQ ID NO: 1; (16) amino acid 26 to 282 of SEQ ID NO: 19corresponding to a mature OMP-1-16 protein encoded by nucleotide16861-17634 of SEQ ID NO: 1; (17) amino acid 26 to 272 of SEQ ID NO: 20corresponding to a mature OMP-1-17 protein encoded by nucleotide18479-19222 of SEQ ID NO: 1; (18) amino acid 33 to 282 of SEQ ID NO: 21corresponding to a mature OMP-1-18 protein encoded by nucleotide19558-20310 of SEQ ID NO: 1; or (19) amino acid 24 to 282 of SEQ ID NO:22 corresponding to a mature OMP-1-19 protein encoded by nucleotide21188-21967 of SEQ ID NO: 1. Excluded from the isolated polypeptidesequence are the sequence SEQ ID NO: 128, 130, 132, 134, 136; or anyfragment thereof. Each EE polypeptide has a specific binding affinityfor an anti-E. ewingii antibody.

In some embodiments, the functional derivative of the EE proteincomprises a sequence which is at least 85%, 90%, 95%, or 98% identicalto one of the sequences (1)-(19), described above.

In some embodiments, the functional derivative comprises animmunoreactive fragment that has a length of from 6 amino acids to lessthan the full length of the EE protein and comprises at least 6consecutive amino acids of one or more sequences selected from the groupconsisting of: (1) SEQ ID NO: 137-155; (2) SEQ ID NO: 156-173; (3) SEQID NO: 174-191; (4) SEQ ID NO: 192-208; (5) SEQ ID NO: 209-227; and (6)any combination of the sequences (1)-(5). Each immunoreactive fragmenthas a specific binding affinity for an anti-E. ewingii antibody.

In some embodiments, the EE polypeptide comprises a sequence selectedfrom the group consisting of (1) SEQ ID NO: 3 corresponding to animmature OMP-1-1 protein; (2) SEQ ID NO: 4 corresponding to an immatureOMP-1-2 protein; (3) SEQ ID NO: 6 corresponding to an immature OMP-1-3protein; (4) SEQ ID NO: 7 corresponding to an immature OMP-1-4 protein;(5) SEQ ID NO: 8 corresponding to an immature OMP-1-5 protein; (6) SEQID NO: 9 corresponding to an immature OMP-1-6 protein; (7) SEQ ID NO: 10corresponding to an immature OMP-1-7 protein; (8) SEQ ID NO: 11correspoding to an immature OMP-1-8 protein; (9) SEQ ID NO: 12corresponding to an immature OMP-1-9 protein; (10) SEQ ID NO: 13corresponding to an immature OMP-1-10 protein; (11) SEQ ID NO: 14corresponding to an immature OMP-1-11 protein; (12) SEQ ID NO: 15corresponding to an immature OMP-1-12 protein; (13) SEQ ID NO: 16corresponding to an immature OMP-1-13 protein; (14) SEQ ID NO: 17corresponding to an immature OMP-1-14 protein; (15) SEQ ID NO: 18corresponding to an immature OMP-1-15 protein; (16) SEQ ID NO: 19corresponding to an immature OMP-1-16; (17) SEQ ID NO: 20 correspondingto an immature OMP-1-17 protein; (18) SEQ ID NO: 21corresponding to animmature OMP-1-18 protein; and (19) SEQ ID NO: 22 corresponding to animmature OMP-1-19 protein.

Also provided herein is an isolated polynucleotide encoding an E.ewingii (EE) protein, or a functional derivative thereof. The EE proteinis selected from the group consisting of: (1) a mature OMP-1-1 proteinencoded by nucleotide 672-1484 of SEQ ID NO: 1; (2) a mature OMP-1-2protein encoded by nucleotide 2116-2871 of SEQ ID NO: 1; (3) a matureOMP-1-3 protein encoded by nucleotide 3610-4395 of SEQ ID NO: 1; (4) amature OMP-1-4 protein encoded by nucleotide 4486-5286 of SEQ ID NO: 1;(5) a mature OMP-1-5 protein encoded by nucleotide 5380-6129 of SEQ IDNO: 1; (6) a mature OMP-1-6 protein encoded by nucleotide 6216-7040 ofSEQ ID NO: 1; (7) a mature OMP-1-7 protein encoded by nucleotide7145-7921 of SEQ ID NO: 1; (8) a mature OMP-1-8 protein encoded bynucleotide 8032-8679 of SEQ ID NO: 1; (9) a mature OMP-1-9 proteinencoded by nucleotide 8772-9536 of SEQ ID NO: 1; (10) a mature OMP-1-10protein encoded by nucleotide 9620-10387 of SEQ ID NO: 1; (11) a matureOMP-1-11 protein encoded by nucleotide 10477-11268 of SEQ ID NO: 1; (12)a mature OMP-1-12 protein encoded by nucleotide 11370-12188 of SEQ IDNO: 1; (13) a mature OMP-1-13 protein encoded by nucleotide 12292-13113of SEQ ID NO: 1; (14) a mature OMP-1-14 protein encoded by nucleotide14530-15312 of SEQ ID NO: 1; (15) a mature OMP-1-15 protein encoded bynucleotide 15689-16450 of SEQ ID NO: 1; (16) a mature OMP-1-16 proteinencoded by nucleotide 16861-17634 of SEQ ID NO: 1; (17) a matureOMP-1-17 protein encoded by nucleotide 18479-19222 of SEQ ID NO: 1; (18)a mature OMP-1-18 protein encoded by nucleotide 19558-20310 of SEQ IDNO: 1; and (19) a mature OMP-1-19 protein encoded by nucleotide21188-21967 of SEQ ID NO: 1. The functional derivative should not havethe sequence SEQ ID NO: 128, 130, 132, 134, 136; or any fragmentthereof; and each functional derivative should have a specific bindingaffinity for an anti-E. ewingii antibody.

In some embodiments, the functional derivative encoded by thepolynucleotide comprises a sequence which is at least 85%, 90%, 95% or98% identical to the sequence (1)-(19), as described above.

In some embodiments, the functional derivative encoded by thepolynucleotide comprises an immunoreactive fragment that has a length offrom 6 amino acids to less than the full length of the EE protein andcomprises: 6 or more consecutive amino acids from the followingsequences: (1) SEQ ID NO: 137-155; (2) SEQ ID NO: 156-173; (3) SEQ IDNO: 174-191; (4) SEQ ID NO: 192-208; (5) SEQ ID NO: 209-227; or (6) anycombination of the sequences (1)-(5); wherein each immunoreactivefragment has a specific binding affinity for an anti-E. ewingiiantibody.

In other embodiments, the EE protein encoded by the polynucleotidecomprises a sequence selected from the group consisting of: (1) SEQ IDNO: 3 corresponding to an immature OMP-1-1 protein; (2) SEQ ID NO: 4corresponding to an immature OMP-1-2 protein; (3) SEQ ID NO: 6corresponding to an immature OMP-1-3 protein; (4) SEQ ID NO: 7corresponding to an immature OMP-1-4 protein; (5) SEQ ID NO: 8corresponding to an immature OMP-1-5 protein; (6) SEQ ID NO: 9corresponding to an immature OMP-1-6 protein; (7) SEQ ID NO: 10corresponding to an immature OMP-1-7 protein; (8) SEQ ID NO: 11corresponding to an immature OMP-1-8 protein; (9) SEQ ID NO: 12corresponding to an immature OMP-1-9 protein; (10) SEQ ID NO: 13corresponding to an immature OMP-1-10 protein; (11) SEQ ID NO: 14corresponding to an immature OMP-1-11 protein; (12) SEQ ID NO: 15corresponding to an immature OMP-1-12 protein; (13) SEQ ID NO: 16corresponding to an immature OMP-1-13 protein; (14) SEQ ID NO: 17corresponding to an immature OMP-1-14 protein; (15) SEQ ID NO: 18corresponding to an immature OMP-1-15 protein; (16) SEQ ID NO: 19corresponding to an immature OMP-1-16; (17) SEQ ID NO: 20 correspondingto an immature OMP-1-17 protein; (18) SEQ ID NO: 21 corresponding to animmature OMP-1-18 protein; (19) SEQ ID NO: 22 corresponding to animmature OMP-1-19 protein.

In one embodiment, the polynucleotide comprises a portion of thenucleotide sequence SEQ ID NO: 1.

The invention also relates to a kit for detecting antibodies specificfor E. ewingii (EE), the kit comprising an isolated EE polypeptide asdescribed herein.

Also provided herein is a method for detecting antibodies specific forE. ewingii (EE). The method includes: (a) contacting a test sample withone or more isolated EE polypeptides, as described herein, underconditions that allow polypeptide/antibody complexes to form; and (b)assaying for the formation of a complex between antibodies in the testsample and the one or more EE polypeptides; wherein the formation of thecomplex is an indication that antibodies specific for E. ewingii arepresent in the test sample.

Also provided are isolated antibodies having specific binding affinityto an isolated EE polypeptide, as described herein.

Also provided is an immunogenic composition comprising one or moreisolated E. ewingii OMP proteins, or immunogenic fragments or variantsthereof, or a fusion protein containing same, and a pharmaceuticallyacceptable carrier. Such a composition is capable of producingantibodies specific to E. ewingii in a subject to whom the immunogeniccomposition has been administered. The isolated E. ewingii OMP proteinfor use in such a composition is selected from the group consisting of:(1) amino acid 24 to 293 of SEQ ID NO: 3 corresponding to a matureOMP-1-1 protein; (2) amino acid 22 to 272 of SEQ ID NO: 4 correspondingto a mature OMP-1-2 protein; (3) amino acid 24 to 284 of SEQ ID NO: 6corresponding to a mature OMP-1-3 protein; (4) amino acid 28 to 293 ofSEQ ID NO: 7 corresponding to a mature OMP-1-4 protein; (5) amino acid24 to 272 of SEQ ID NO: 8 corresponding to a mature OMP-1-5 protein; (6)amino acid 26 to 299 of SEQ ID NO: 9 corresponding to a mature OMP-1-6protein; (7) amino acid 27 to 284 of SEQ ID NO: 10 corresponding to amature OMP-1-7 protein; (8) amino acid 29 to 243 of SEQ ID NO: 11corresponding to a mature OMP-1-8 protein; (9) amino acid 28 to 281 ofSEQ ID NO: 12 corresponding to a mature OMP-1-9 protein; (10) amino acid26 to 280 of SEQ ID NO: 13 corresponding to a mature OMP-1-10 protein;(11) amino acid 28 to 290 of SEQ ID NO: 14 corresponding to a matureOMP-1-11 protein; (12) amino acid 27 to 298 of SEQ ID NO: 15corresponding to a mature OMP-1-12 protein; (13) amino acid 30 to 302 ofSEQ ID NO: 16 corresponding to a mature OMP-1-13 protein; (14) aminoacid 26 to 285 of SEQ ID NO: 17 corresponding to a mature OMP-1-14protein; (15) amino acid 26 to 278 of SEQ ID NO: 18 corresponding to amature OMP-1-15 protein; (16) amino acid 26 to 282 of SEQ ID NO: 19corresponding to a mature OMP-1-16 protein; (17) amino acid 26 to 272 ofSEQ ID NO: 20 corresponding to a mature OMP-1-17 protein; (18) aminoacid 33 to 282 of SEQ ID NO: 21 corresponding to a mature OMP-1-18protein; and (19) amino acid 24 to 282 of SEQ ID NO: 22 corresponding toa mature OMP-1-19 protein.

In one embodiment, the fusion protein in such a composition comprises anisolated E. ewingii OMP protein, or immunogenic fragment or variantthereof, and an N-terminal or C-terminal peptide or tag.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Strategy of E. ewingii omp-1 cluster sequencing. E. chaffeensisomp-1 and E. canis p30 were aligned to design 21 pairs of degenerateprimers. The OMP-1 multigene locus was divided into three fragments eachcomposed of seven shorter fragments (A). The initial nested touch-downPCRs generated four specific sequences within fragments 1 and 2 (B). Twofragments were amplified by nested touchdown PCR within fragment 3 usingthe p28-19 sequence and degenerate primers. Specific primers weredesigned to close all gaps (C). Two poly A/T and G/C regions were clonedinto a TA vector and sequenced. The final sequence (24,126 bp) wasassembled using SeqMan program in the DNASTAR software.

FIG. 2. Schematic representation of the organization of the E. ewingiiomp-1 gene cluster. Genes are represented as boxes with arrowsindicating their orientation. omp-1s are shown in a horizontal shadingpattern. Black, white, and gray boxes show tr1, unknown genes, and secA,respectively.

FIG. 3. Synteny analysis of the E. ewingii (Ee) omp-1 cluster relativeto E. chaffeensis (Ech), E. canis (Eca), and E. ruminantium (Eru) by theArtemis comparison tool.

FIG. 4. Dot plot analysis of the E. ewingii omp-1 cluster (A), and theE. ewingii omp-1 cluster relative to E. chaffeensis (B), E. canis (C),or E. ruminantium (D). Repetitive regions consisting of multiplehomologous DNA segments were analyzed using the web-based dot plotprogram (JDotter) (available at athena.bioc.uvic.ca/index.php. Thewindow cutoff was set to the default. The α, β and γ repetitive regionsdescribed by Ohashi et al., 2001, Infect Immun 69:2083-2091, are markedby lines.

FIG. 5. Phylogram of OMP proteins of E. ewingii, E. chaffeensis, E.canis, and E. ruminantium. A total of 39 OMPs were segregated into 10clusters with four or three Ehrlichia species each, but 40 remainingproteins were not. The tree was constructed using the Neighbor-Joining(NEIGHBOR program from PHYLIP) method based on the alignment generatedwith CLUSTAL V; 1000 bootstrap replications were performed. The nodessupported by bootstrap values greater than 60% are labeled. The OMPsencoded by the three repetitive regions in FIG. 4 are indicated by α,β1, and β2. Branch lengths are proportional to percent divergence. Thecalibration bar is on the lower left.

FIG. 6. ELISA analysis of E. ewingii- and E. chaffeensis-infected dogswith the 19 EEOMP-1 oligopeptides. Preinfection control andpost-infection plasma from dogs were allowed to react with the 19synthesized EEOMP-1 specific peptides. The y-axis showsOD_(405nm)-OD_(492nm). A reaction was considered to be positive when theplasma from infected dogs yielded an OD_(405nm)-OD_(492nm) value greaterthan the mean OD_(405nm)-OD_(492nm) of preinfection control plasma+threestandard deviations (dashed line with closed triangles). Representativedata of 3-5 assays is shown. Reactivity ratios of E. ewingii/controlplasma (EW/Control) and E. chaffeensis/control plasma (ECH/Control) werecalculated based on the averages of three EW-positive and threeECH-positive samples, respectively, to four negative control samples.EEOMP-1 peptides that showed good sensitivity and specificity fordetecting E. ewingii infection are underlined.

FIG. 7. Alignment of the 19 E. ewingii OMP-1 proteins.

FIG. 8. Alignment of E. ewingii OMP-1, E. canis P30, E. chaffeensisOMP-1, and E. ruminantium MAP1 proteins.

DETAILED DESCRIPTION

The present invention will now be described with occasional reference tosome specific embodiments disclosed herein. This invention may, however,be embodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription herein is for describing particular embodiments only and isnot intended to be limiting. As used in the description and the appendedclaims, the singular forms “a,” “an,” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise. All publication, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope are approximations,the numerical values set forth in the specific examples are reported asprecisely as possible. Any numerical values, however, inherently containcertain errors necessarily resulting from error found in theirrespective measurements.

DEFINITIONS

A “polynucleotide” or “nucleic acid molecule,” as is generallyunderstood and used herein, refers to a polymer of nucleotides, andincludes but should not be limited to DNA and RNA.

“Recombinant DNA” is any DNA molecule formed by joining DNA segmentsfrom different sources and produced using “recombinant DNA” technology(also known as “molecular genetic engineering”).

A “DNA segment or fragment,” as is generally understood and used herein,refers to a molecule comprising a linear stretch of nucleotides whereinthe nucleotides are present in a sequence that can encode, through thegenetic code, a molecule comprising a linear sequence of amino acidresidues that is referred to as a protein, a protein fragment or apolypeptide.

“Gene” refers to a DNA sequence related to a single polypeptide chain orprotein, and as used herein includes the 5′ and 3′ untranslated ends.The polypeptide can be encoded by a full-length sequence or any portionof the coding sequence, so long as the functional activity (i.e.immuno-reactivity or immunogenicity) of the protein is retained.

“Complementary,” as used herein, refers to the natural binding ofpolynucleotides under permissive salt and temperature conditions by basepairing.

“Complementary DNA” or “cDNA” refers to recombinant nucleic acidmolecules synthesized by reverse transcription of messenger RNA(“mRNA”).

Open Reading Frame (“ORF”). A series of codons (base triplets) which canbe translated into a protein without any termination codons interruptingthe relevant reading frames. An ORF can be evidence that a DNA sequenceis part of a gene.

Restriction Endonuclease. A “restriction endonuclease” (also“restriction enzyme”) is an enzyme that has the capacity to recognize aspecific base sequence (usually 4, 5, or 6 base pairs in length) in aDNA molecule, and to cleave the DNA molecule at every place where thissequence appears. For example, EcoRI recognizes the base sequenceGAATTC/CTTAAG.

Restriction Fragment. The DNA molecules produced by digestion with arestriction endonuclease are referred to as “restriction fragments.” Anygiven genome can be digested by a particular restriction endonucleaseinto a discrete set of restriction fragments.

Agarose Gel Electrophoresis. To determine the length of restrictionfragments, an analytical method for fractionating double-stranded DNAmolecules on the basis of size is required. The most commonly usedtechnique (though not the only one) for achieving such a fractionationis “agarose gel electrophoresis.” The principle of this method is thatDNA molecules migrate through the gel as though it were a sieve thatretards the movement of the largest molecules to the greatest extent andthe movement of the smallest molecules to the least extent. Note thatthe smaller the DNA fragment, the greater the mobility underelectrophoresis in the agarose gel.

The DNA fragments fractionated by “agarose gel electrophoresis” can bevisualized directly by a staining procedure if the number of fragmentsincluded in the pattern is small. The DNA fragments of genomes can bevisualized successfully. However, most genomes, including the humangenome, contain far too many DNA sequences to produce a simple patternof restriction fragments. For example, the human genome is digested intoapproximately 1,000,000 different DNA fragments by EcoRI. In order tovisualize a small subset of these fragments, a methodology referred toas the Southern hybridization procedure can be applied.

Southern Transfer Procedure. The purpose of the “Southern transferprocedure” (also “Southern blotting”) is to physically transfer DNAfractionated by agarose gel electrophoresis onto a nitrocellulose filterpaper or another appropriate surface or method, while retaining therelative positions of DNA fragments resulting from the fractionationprocedure. The methodology used to accomplish the transfer from agarosegel to nitrocellulose involves drawing the DNA from the gel into thenitrocellulose paper by capillary action or electrophoretic transfer.

Nucleic Acid Hybridization. “Nucleic acid hybridization” depends on theprinciple that two single-stranded nucleic acid molecules that havecomplementary base sequences will reform the thermodynamically favoreddouble-stranded structure if they are mixed under the proper conditions.The double-stranded structure will be formed between two complementarysingle-stranded nucleic acids even if one is immobilized on anitrocellulose filter. In the Southern hybridization procedure, thelatter situation occurs. As noted previously, the DNA of the test sampleto be tested is digested with a restriction endonuclease, fractionatedby agarose gel electrophoresis, converted to the single-stranded form,and transferred to nitrocellulose paper, making it available forreannealing to the hybridization probe. Examples of hybridizationconditions can be found in Ausubel, F. M. et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., New York, N.Y. (1989). Forexample, a nitrocellulose filter is incubated overnight at 68° C. withlabeled probe in a solution containing 50% formamide, high salt (either5×SSC[20×: 3M NaCl/0.3M trisodium citrate] or 5×SSPE [20×: 3.6MNaCl/0.2M NaH₂PO₄/0.02M EDTA, pH 7.7]), 5×Denhardt's solution, 1% SDS,and 100 μg/ml denatured salmon sperm DNA. This is followed by severalwashes in 0.2×SSC/0.1% SDS at a temperature selected based on thedesired stringency: room temperature (low stringency), 42° C. (moderatestringency) or 68° C. (high stringency). The temperature selected isdetermined based on the melting temperature (Tm) of the DNA hybrid.

Hybridization Probe. To visualize a particular DNA sequence in theSouthern hybridization procedure, a labeled DNA molecule or“hybridization probe” is reacted to the fractionated DNA bound to thenitrocellulose filter. The areas on the filter that carry DNA sequencescomplementary to the labeled DNA probe become labeled themselves as aconsequence of the reannealing reaction. The areas of the filter thatexhibit such labeling are visualized. The hybridization probe isgenerally produced by molecular cloning of a specific DNA sequence.

A “purified” or “isolated” polypeptide or nucleic acid is a polypeptideor nucleic acid that has been separated from a cellular component.Purified or isolated polypeptides or nucleic acids have been purified toa level of purity not found in nature.

A “mutation” is any detectable change in the genetic material which canbe transmitted to daughter cells and possibly even to succeedinggenerations giving rise to mutant cells or mutant individuals. If thedescendants of a mutant cell give rise only to somatic cells inmulticellular organisms, a mutant spot or area of cells arises.“Mutations” in the germ line of sexually reproducing organisms can betransmitted by the gametes to the next generation resulting in anindividual with the new mutant condition in both its somatic and germcells. A “mutation” can be any (or a combination of) detectable,unnatural change affecting the chemical or physical constitution,mutability, replication, phenotypic function, or recombination of one ormore deoxyribonucleotides; nucleotides can be added, deleted,substituted for, inverted, or transposed to new positions with andwithout inversion. “Mutations” can occur spontaneously and can beinduced experimentally by application of mutagens.

Oligonucleotide or Oligomer. A molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three. Itsexact size will depend on many factors, which in turn depend on theultimate function or use of the “oligonucleotide.” An “oligonucleotide”can be derived synthetically or by cloning.

Sequence Amplification. A method for generating large amounts of atarget sequence. In general, one or more amplification primers areannealed to a nucleic acid sequence. Using appropriate enzymes,sequences found adjacent to, or in between the primers are amplified.

Amplification Primer or “Primer”. An oligonucleotide which is capable ofannealing adjacent to a target sequence and serving as an initiationpoint for DNA synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is initiated.

Vector. A plasmid or phage DNA or other DNA sequence into which DNA canbe inserted to be cloned. The “vector” can replicate autonomously in ahost cell, and can be further characterized by one or a small number ofendonuclease recognition sites at which such DNA sequences can be cut ina determinable fashion and into which DNA can be inserted. The “vector”can further contain a marker suitable for use in the identification ofcells transformed with the “vector”. Markers, for example, aretetracycline resistance or ampicillin resistance. The words “cloningvehicle” are sometimes used for “vector.”

Expression. “Expression” is the process by which a structural geneproduces a polypeptide. It involves transcription of the gene into mRNA,and the translation of such mRNA into polypeptide(s).

Expression Vector. A vector or vehicle similar to a cloning vector butwhich is capable of expressing a gene which has been cloned into it,after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences.

Expression control sequences will vary depending on whether the vectoris designed to express the operably linked gene in a prokaryotic oreukaryotic host and can additionally contain transcriptional elementssuch as enhancer elements, termination sequences, tissue-specificityelements, and/or translational initiation and termination sites.

Embodiments

The omp-1 gene cluster sequence of E. ewingii (SEQ ID NO: 1) contains 23open reading frames (ORFs), as outlines in Table 1. The ORFs encode E.ewingii (EE) outer membrane proteins (OMPs) OMP-1-1, OMP-1-2, OMP-1-3,OMP-1-4, OMP-1-5, OMP-1-6, OMP-1-7, OMP-1-8, OMP-1-9, OMP-1-10,OMP-1-11, OMP-1-12, OMP-1-13, OMP-1-14, OMP-1-15, OMP-1-16, OMP-1-17,OMP-1-18, OMP-1-19, as well as the two proteins UN2 and UN3. As usedherein, the term “EE proteins” and “EE OMPs” are used interchangeablyand refer to the above mentioned 19 EE OMPs of E. ewingii in theirmature (i.e. lacking the signal peptide) as well as immature (i.e.including the signal peptide) forms, as listed in Table 1.

“EE polypeptides” include EE proteins and functional derivatives of theEE proteins, as well as fusion proteins made from such proteins or theirfunctional derivatives. The invention also relates to isolatedpolynucleotides encoding EE polypeptides, probes, primers, antibodiesand methods of their production, compositions containing one or more ofthe above mentioned molecules, as well as methods of using the probes,primers, EE polypeptides and antibodies for the purpose of diagnosis,screening, therapy and production of vaccines against E. ewingii.

Isolated E. Ewingii Polypeptides

The 24-kb omp-1 gene locus contains 23 open reading frames (ORFs),numbered ORF 1 to 23 (see Table 1). These 23 ORFs are arranged in tandemexcept for three ORFs (ORF 19, 20, and 21) that are in the oppositeorientation (i.e. the complementary strand encodes the OMP-1-17,OMP-1-18 and OMP-1-19 proteins, respectively). Nineteen of these 23 ORFsencode outer membrane proteins (EE proteins) enumerated as E. ewingii(EE)OMP-1-1 to EEOMP-1-19. Two ORFs encode the proteins UN2 and UN3. Themature OMP-1 proteins of E. ewingii have a molecular mass of about 25.1to 31.3 kDa; and isoelectric points of 5.03 to 9.80. The properties ofthe polypeptides encoded by the ORFs of the E. ewingii omp-1 genecluster, including signal peptide lengths, molecular mass, andisoelectric points of mature proteins, as well as the sequenceidentifiers for each protein, are shown in Table 1.

TABLE 1 Properties of E. ewingii proteins Length (bp) Upstream (based onthe signal SEQ intergenic omp-1 nucleotide peptide Molecular OMP-1 ORFID space sequence AA AA Mass^(a) number number NO (bp) SEQ ID NO: 1)number number (Da) PI^(a) NA ORF-1 2 NA 357(3-359) 118 NA 13687.9^(b)7.93^(b) Hypothetical transcriptional regulator OMP-1-1 ORF-2 3 244882(603-1484) 293 23 29848.6 6.38 OMP-1-2 ORF-3 4 568 819(2053-2871) 27221 28125.9 8.61 NA ORF-4 5 101 558(2973-3530) 185 32 18000.7 9.80 UN2OMP-1-3 ORF-5 6 10 855(3541-4395) 284 23 29372.6 7.07 OMP-1-4 ORF-6 7 6882(4405-5286) 293 27 30194.6 9.47 OMP-1-5 ORF-7 8 24 819(5311-6129) 27223 28174.6 5.39 OMP-1-6 ORF-8 9 11 900(6141-7040) 299 25 30194.6 9.47OMP-1-7 ORF-9 10 26 855(7067-7921) 284 26 28864.7 5.43 OMP-1-8 ORF-10 1126 732(7948-8679) 243 28 25098.4 7.88 OMP-1-9 ORF-11 12 11846(8691-9536) 281 27 28661.4 7.24 OMP-1-10 ORF-12 13 8 843(9545-10387)280 25 28476.4 6.25 OMP-1-11 ORF-13 14 8 867(10396-11268) 290 27 29488.95.90 OMP-1-12 ORF-14 15 23 897(11292-12188) 298 26 30036.8 5.89 OMP-1-13ORF-15 16 16 909(12205-13113) 302 29 31275.1 5.31 OMP-1-14 ORF-16 171343 858(14455-15312) 285 25 28864.7 5.03 OMP-1-15 ORF-17 18 301837(15614-16450) 278 25 28862.8 5.58 OMP-1-16 ORF-18 19 335849(16786-17634) 282 25 28476.0 5.41 OMP-1-17 ORF-19 20 769819(18404-19222) 272 25 27782.3 6.50 OMP-1-18 ORF-20 21 539849(19462-20310) 282 32 28603.4 5.08 OMP-1-19 ORF-21 22 808849(21119-21967) 282 23 30505.0 7.88 NA ORF-22 23 1082 408(23050-23457)135 NA 14800.0 3.85 NA ORF-23 24 390 285(23848-24126) 93 NA 10533.3^(b)9.40^(b)

In one aspect, the invention relates to isolated polypeptides comprisingan amino acid sequence corresponding to EE proteins, or functionalderivatives thereof. The isolated polypeptides of the inventionexpressly exclude a peptide that is the 505-bp E. ewingii p28-1 peptidedeposited in GenBank accession numbers: AF287961 (SEQ ID NOS 127-128),AF287962 (SEQ ID NOS 129-130), AF287963 (SEQ ID NOS 131-132), AF287964(SEQ ID NOS 133-134, AF287966 (SEQ ID NOS 135-136); or any fragmentthereof.

The EE proteins include the immature (i.e. with the signal peptide) aswell as the mature form (lacking the signal peptide) proteins of E.Ewingii, as described below and in Table 1.

In one embodiment, the polypeptide comprises the sequence of an immatureOMP-1-1 protein having the amino acid sequence SEQ ID NO: 3, encoded bynucleotide 603-1484 of SEQ ID NO: 1. In another embodiment, thepolypeptide comprises the sequence of a mature OMP-1-1 protein havingthe amino acid sequence from residue 24 to 293 of SEQ ID NO: 3, encodedby nucleotide 672-1484 of SEQ ID NO: 1.

In one embodiment, the polypeptide comprises the sequence of an immatureOMP-1-2 protein having the amino acid sequence SEQ ID NO: 4, encoded bynucleotide 2053-2871 of SEQ ID NO: 1. In another embodiment, thepolypeptide comprises the sequence of a mature OMP-1-2 protein havingthe amino acid sequence from residue 22 to 272 of SEQ ID NO: 4, encodedby nucleotide 2116-2871 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-3 protein having the amino acid sequence SEQ ID NO: 6,encoded by nucleotide 3541-4395 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-3 proteinhaving the amino acid sequence from residue 24-284 of SEQ ID NO: 6,encoded by nucleotide 3610-4395 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-4 protein having the amino acid sequence SEQ ID NO: 7,encoded by nucleotide 4405-5286 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-2 proteinhaving the amino acid sequence from residue 28 to 293 of SEQ ID NO: 7,encoded by nucleotide 4486-5286 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-5 protein having the amino acid sequence SEQ ID NO: 8,encoded by nucleotide 5311-6129 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-5 proteinhaving the amino acid sequence from residue 24 to 272 of SEQ ID NO: 8,encoded by nucleotide 5380-6129 of SEQ ID NO: 1.

In another embodiment the polypeptide comprises the sequence of animmature OMP-1-6 protein having the amino acid sequence SEQ ID NO: 9,encoded by nucleotide 6141-7040 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-6 proteinhaving the amino acid sequence from residue 26 to 299 of SEQ ID NO: 9,encoded by nucleotide 6216-7040 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-7 protein having the amino acid sequence SEQ ID NO: 10,encoded by nucleotide 7067-7921 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-7 proteinhaving the amino acid sequence from residue 27 to 284 of SEQ ID NO: 10,encoded by nucleotide 7145-7921 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-8 protein having the amino acid sequence SEQ ID NO: 11,encoded by nucleotide 7948-8679 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-8 proteinhaving the amino acid sequence from residue 29 to 243 of SEQ ID NO: 11,encoded by nucleotide 8032-8679 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-9 protein having the amino acid sequence SEQ ID NO: 12,encoded by nucleotide 8691-9536 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-9 proteinhaving the amino acid sequence from residue 28 to 281 of SEQ ID NO: 12,encoded by nucleotide 8772-9536 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-10 protein having the amino acid sequence SEQ ID NO: 13,encoded by nucleotide 9545-10387 of SEQ ID NO: 1. In another embodiment,the polypeptide comprises the sequence of a mature OMP-1-10 proteinhaving the amino acid sequence from residue 26 to 280 of SEQ ID NO: 13.encoded by nucleotide 9620-10387 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-11 protein having the amino acid sequence SEQ ID NO: 14,encoded by nucleotide 10396-11268 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-11protein having the amino acid sequence from residue 28 to 290 of SEQ IDNO: 14, encoded by encoded by nucleotide 10477-11268 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-12 protein having the amino acid sequence SEQ ID NO: 15,encoded by nucleotide 11292-12188 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-12protein having the amino acid sequence from residue 27 to 298 of SEQ IDNO: 15, encoded by nucleotide 11370-12188 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-13 protein having the amino acid sequence SEQ ID NO: 16,encoded by nucleotide 12205-13113 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-13protein having the amino acid sequence from residue 30 to 302 of SEQ IDNO: 16, encoded by nucleotide 12292-13113 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-14 protein having the amino acid sequence SEQ ID NO: 17,encoded by nucleotide 14455-15312 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-14protein having the amino acid sequence from residue 26 to 285 of SEQ IDNO: 17, encoded by nucleotide 14530-15312 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-15 protein having the amino acid sequence SEQ ID NO: 18,encoded by nucleotide 15614-16450 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-15protein having the amino acid sequence from residue 26 to 278 of SEQ IDNO: 18, encoded by nucleotide 15689-16450 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-16 protein having the amino acid sequence SEQ ID NO: 19,encoded by nucleotide 16786-17634 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-16protein having the amino acid sequence from residue 26 to 282 of SEQ IDNO: 19, encoded by nucleotide 16861-17634 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-17 protein having the amino acid sequence SEQ ID NO: 20,encoded by nucleotide 18404-19222 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-17protein having the amino acid sequence from residue 26 to 272 of SEQ IDNO: 20, encoded by nucleotide 18479-19222 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-18 protein having the amino acid sequence SEQ ID NO: 21,encoded by nucleotide 19462-20310 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-18protein having the amino acid sequence from residue 33 to 282 of SEQ IDNO: 21, encoded by nucleotide 19558-20310 of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises the sequence of animmature OMP-1-19 protein having amino acid sequence SEQ ID NO: 22,encoded by nucleotide 21119-21967 of SEQ ID NO: 1. In anotherembodiment, the polypeptide comprises the sequence of a mature OMP-1-19protein having the amino acid sequence from residue 24 to 282 of SEQ IDNO: 22, encoded by nucleotide 21188-21967 of SEQ ID NO: 1.

Each EE OMP-1 protein has several conserved regions, with amino acidsequences that are more or less conserved between the nineteen EE OMP-1proteins. Each EE OMP-1 protein also includes 4 variable loops, referredto hereinafter as loops 1-4, that have variable amino sequences betweenthe nineteen EE OMP-1 proteins (FIG. 7). Tables 2-5 shows that aminoacid sequence of each of the variable loops in the EE OMP-1 proteins.

TABLE 2 Sequences of variable region loop 1 in EE OMP-1-1 to OMP-1-19From aa residue OMP # Loop 1 SEQ ID NO: # to aa residue # OMP1-1SPIPIDFSNESEMV 137 24 to 37 OMP1-2 QGLNDNIFKN 138 22 to 31 OMP1-3LLALENNLSGGVGHI 139 21-31 OMP1-4 SFVFSMQGRSNIT 140 23-35 OMP1-5VLIDTTEKDYASNV 141 24-37 OMP1-6 AISIDNNIIDQNL 142 25-37 OMP1-7PISNNSEDNIF 143 28-38 OMP1-8 LNNAEDHKD 144 31-39 OMP1-9 ESNHYDKSL 14528-36 OMP1-10 EVITHNDNKHPGI 146 26-38 OMP1-11 AKNNYSYINPVL 147 27-38OMP1-12 ETTIINQPSGL 148 27-37 OMP1-13 ETIVDDIDRQFRL 149 30-42 OMP1-14ADPMNSNDVSINDSKE 150 25-40 OMP1-15 LVSFIPCI 151 15-22 OMP1-16 FICELPGV152 15-22 OMP1-17 DVVVSEEKR 153 26-34 OMP1-18 FYASMSFGMSNTLANQVSPIS 15419-39 OMP1-19 LVSDASDSHTKSVSL 155 26-34

TABLE 3 Sequences of variable region loop 2 in EE OMP-1-1 to OMP-1-19SEQ From aa residue OMP # Loop 2 ID NO: # to aa residue # OMP1-1YKSTGNSEADKSEKELTLFTLKESTQAPDFTKKET 156 59-93 OMP1-2SKDTIGIFALKKDASLPTDIKKNS 157 54-77 OMP1-3 MEEATIGAVIPKSLKQDAEDITLSILALST158 53-82 OMP1-4 FDTKDPIGLIRSARSTEPSVLKINTH 159 60-85 OMP1-5SKTKNSIALEKPIESNSNILKS 160 60-81 OMP1-6 KKVDLIALKNDVTHITEEILKDP 16162-84 OMP1-7 FATQKLMRVKKDSKEGLPNILKSKD 162 63-87 OMP1-8CIIRLITVKDSHFFSINTSSYNLCLEKHKNDI 163 54-85 OMP1-9DINTKGLFKLGHGVTLVEEDIKNHLQ 164 58-83 OMP1-10 ATTVQLGLNYTAAPIDDIKTSSK 16561-84 OMP1-11 HYDTQLLELKKEVGSVTNTVIQAYANYNVPSQAP 166 60-94 OMP1-12VATKHLIALKKSVDSINAEKATPHNQGLGKPD 167 60-91 OMP1-13VTTKYLTALKKDADPTEKTGSTPHEKGLGKPD 168 65-96 OMP1-14PIEGAISPTKKVLGLNKGGSIANSHDFSKIDP 169 64-95 OMP1-15DTIETIATFGLSKTYNRSSPIHSDFTDKS 170 58-86 OMP1-16 — — — OMP1-17IPGLTKKIFALSYDATDITKETSFKQA 171 58-84 OMP1-18QILHDVATERVVGLKHDLLESADKLVDNLYNFDLSED 172 60-96 OMP1-19LTSGIIANKRVLGLKNDILINADEAIKNLS 173 61-90

TABLE 4 Sequences of variable region loop 3 in EE OMP-1-1 to OMP-1-19SEQ From aa residue OMP # Loop 3-1 ID NO # to aa residue # OMP1-1DKQKHTHPDNH 174 136-146 OMP1-2 EGYTKITGVEQH 175 122-132 OMP1-3VSAPSGYDDNYAYSI 176 123-139 OMP1-4 IKRLVNYASRDGH 177 129-141 OMP1-5ELNSSSLISSNNHYTQLYE 178 126-144 OMP1-6 ITDCSNCTIN 179 127-136 OMP1-7KDPKDCSVKDAFRHL 180 130-144 OMP1-8 TEDKYLTSEQEVNDY 181 120-134 OMP1-9DLKNCTIQ 182 126-133 OMP1-10 TDPGNYTIK 183 127-135 OMP1-11 KNSGHSSIDAHR184 136-147 OMP1-12 TLNDAF 185 130-135 OMP1-13 TISNAF 186 135-140OMP1-14 KYYGLFREGTPQEEEH 187 146-161 OMP1-15 SNGAHM 188 121-126 OMP1-16— — — OMP1-17 QFYREGSNNYKF 189 123-134 OMP1-18 VQDTKSHIVDDNYR 190121-134 OMP1-19 RDTKNHIIDNN 191 134-144 SEQ From aa residue OMP #Loop 3-2 ID NO: # to aa residue # OMP1-1 SCTEQEMKPAQQNGSSKDGN 192151-170 OMP1-2 LDTNGNQPKTDK 193 139-150 OMP1-3 SIEVPQLRSLPYHYT 194138-152 OMP1-4 IRPDTFFNNSIPYAFNA 195 146-162 OMP1-5 ANFQNFATSR 196145-154 OMP1-6 KDNNQVQPKAHDSSSTDSNNSSNNTKKSYFTF 197 148-175 OMP1-7LDTGLSMPKEKK 198 150-161 OMP1-8 VNDYNIISAI 199 131-140 OMP1-9ICKENDKPTPKEKK 200 145-158 OMP1-10 MNSSSNNQPKDKQFT 201 146-160 OMP1-11HSNNGNTQQNPFA 202 154-166 OMP1-12 IESDQNKFQPKNANSNSSNKIYHT 203 154-177OMP1-13 SESSKEPQPKNPNSAGNNKIFHT 204 159-181 OMP1-14 — — — OMP1-15KDNANIGTTPQDKK 205 143-156 OMP1-16 — — — OMP1-17 ETTISKKF 206 141-148OMP1-18 HGPAKHIN 207 152-159 OMP1-19 SKQDNLNSD 208 151-159

TABLE 5 Sequences of variable region loop 4 in EE OMP-1-1 to OMP-1-19SEQ From aa residue OMP # Loop 4 ID NO # to aa residue # OMP1-1TVQYPVKLTSPPTHIDPVVYFHSD 209 258-281 OMP1-2 NYPTDNNTTKTTVSAI 210 241-256OMP1-3 LLDYPSYYRSLTSLSDNDPNRILPF 211 238-262 OMP1-4PLMLSPSTPRRRIPPQSSSEVQDATGLL 212 249-276 OMP1-5 YTQYVSGINSLQEI 213234-247 OMP1-6 TYAYILKDS 214 266-274 OMP1-7 NHVVELDDF 215 251-259 OMP1-8SKIHYAIILSNNKYLQNSLGDTKTNTY 216 208-234 OMP1-9 QNMFDSNE 217 249-256OMP1-10 QHVVTLDT 218 248-255 OMP1-11 QYVNTTTSQAIN 219 254-265 OMP1-12QIIIAELNDA 220 265-273 OMP1-13 QHVAELNDD 221 269-277 OMP1-14KTPVTLDTAPQT 222 252-263 OMP1-15 DITPLKPNGIENTTATHVLV 223 242-256OMP1-16 DIATILPSGSSIKDNQY 224 250-262 OMP1-17 YERVEIAYHPSIEEA 225229-245 OMP1-18 EYSNIPVQYPRNLFYA 226 242-257 OMP1-19QYSSISVKYPKVLVFPSTRS 227 242-261

Also provided herein are functional derivatives of the EE proteinsenumerated above. A “functional derivative” of an EE protein or peptidesequence is a molecule that possesses immunoreactivity to EE antibodiesthat is substantially similar to that of the corresponding EE protein orpeptide, i.e. an “immunoreactive” functional derivative is a polypeptidethat has a specific binding affinity for anti-E.ewingii antibodies. Theterm “specific binding affinity”refers to binding with an affinity forthe EE antibodies that is substantially greater than the bindingaffinity for E. canis, and/or E. chaffeensis, and/or E. ruminantium.

The functional derivatives of an EE protein can be identified using anyof a variety of routine assays for detecting peptide antigen-antibodycomplexes, the presence of which is an indicator of selective binding.Such assays include, without limitation, enzyme-linked immunosorbentassays (ELISA), radioimmunoassays, western blotting, enzymeimmunoassays, flourescence immunoassays, luminescent immunoassays andthe like. Methods for detecting a complex between a peptide and anantibody, and thereby determining if the peptide is an “immunoreactivefunctional derivative” are well known to those skilled in the art andare described, for example, in Example 1, as well as in ANTIBODIES: ALABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring HarborLaboratory Press, 2.sup.nd ed. 1998a); and USING ANTIBODIES: ALABORATORY MANUAL: PORTABLE PROTOCOL No. 1 (Edward Harlow & David Lane,Cold Spring Harbor Laboratory Press, 1998b), which are herebyincorporated by reference in their entirety.

In one embodiment, the “specific binding affinity” of a functionalderivative is defined as an ELISA assay result, where the ratio of E.chaffeensis or E. canis plasma reactivity/control plasma reactivity is˜1.00, and where E. ewingii plasma reactivity yields anOD_(405nm)-OD_(492nm) value greater than the mean OD_(405nm)-OD_(492nm)of preinfection control plasma+three standard deviations.

Thus, the terms “functional derivative” and “immunoreactive functionalderivative” are used intechangeably and refer to peptides and proteinsthat can function in substantially the same manner as the EE proteins orpeptides disclosed herein, and can be substituted for the E. ewingiiproteins or peptides in any aspect of the present invention.

A “functional derivative” of a protein or peptide can containpost-translational modifications such as covalently linked carbohydrate,depending on the necessity of such modifications for the performance ofa specific function. The term “functional derivative” is intended toinclude the immunoreactive “variants” and “fragments” of the EEproteins.

A “variant” of an EE protein refers to a molecule substantially similarin structure and immunoreactivity to the EE protein. Thus, provided thattwo molecules possess a common immunoreactivity and can substitute foreach other, they are considered “variants” as that term is used hereineven if the composition or secondary, tertiary, or quaternary structureof one of the molecules is not identical to that found in the other, orif the amino acid or nucleotide sequence is not identical. Thus, in oneembodiment, a variant refers to a protein whose amino acid sequence issimilar to the amino acid sequences of a mature EE protein, hereinafterreferred to as the reference amino acid sequence, but does not have 100%identity with the respective reference sequence. The variant protein hasan altered sequence in which one or more of the amino acids in thereference sequence is deleted or substitutued, or one or more aminoacids are inserted into the sequence of the reference amino acidsequence. As a result of the alterations, the variant protein has anamino acid sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the referencesequence. For example, variant sequences which are at least 95%identical have no more than 5 alterations, i.e. any combination ofdeletions, insertions or substitutions, per 100 amino acids of thereference sequence. Percent identity is determined by comparing theamino acid sequence of the variant with the reference sequence using anyavailable sequence alignment program. An example includes the MEGALIGNproject in the DNA STAR program. Sequences are aligned for identitycalculations using the method of the software basic local alignmentsearch tool in the BLAST network service (the National Center forBiotechnology Information, Bethesda, Md.) which employs the method ofAltschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990) J. Mol. Biol. 215, 403-410. Identities are calculated by theAlign program (DNAstar, Inc.) In all cases, internal gaps and amino acidinsertions in the candidate sequence as aligned are not ignored whenmaking the identity calculation.

In some embodiments, the variant proteins include all or substantiallyall of the amino acid sequences of the variable loops, presented inTables 2-5. In these embodiments, the amino acid changes are made inareas other than the variable loops of Tables 2-5. Thus, for example,one or more amino acids of the conservative regions located between thevariable loops can be changed without significantly altering theimmunoreactivity of the resultant variant protein.

Variants of the EE proteins can include nonconservative as well asconservative amino acid substitutions. A conservative substitution isone in which the substituted amino acid has similar structural orchemical properties with the corresponding amino acid in the referencesequence. By way of example, conservative amino acid substitutionsinvolve substitution of one aliphatic or hydrophobic amino acids, e.g.alanine, valine, leucine and isoleucine, with another, substitution ofone hydroxyl-containing amino acid, e.g. serine and threonine, withanother, substitution of one acidic residue, e.g. glutamic acid oraspartic acid, with another, replacement of one amide-containingresidue, e.g. asparagine and glutamine, with another, replacement of onearomatic residue, e.g. phenylalanine and tyrosine, with another;replacement of one basic residue, e.g. lysine, arginine and histidine,with another; and replacement of one small amino acid, e.g., alanine,serine, threonine, methionine, and glycine, with another.

The alterations are designed not to abolish the immunoreactivity of thevariant EE protein with antibodies that bind to the reference protein.Guidance in determining which amino acid residues may be substituted,inserted or deleted without abolishing such immunoreactivity of thevariant protein are found using computer programs well known in the art,for example, DNASTAR software.

Preparation of an EE protein variant in accordance herewith can beachieved by site-specific mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the protein. Site-specificmutagenesis allows the production of EE protein variants through the useof specific oligonucleotide sequences that encode the DNA sequence ofthe desired mutation. In general, the technique of site-specificmutagenesis is well known in the art, as exemplified by publicationssuch as Adelman et. al., DNA 2:183 (1983) and Ausubel et al. “CurrentProtocols in Molecular Biology”, J. Wiley & Sons, N.Y., N.Y., 1996. Aswill be appreciated, the site-specific mutagenesis technique can employa phage vector that exists in both a single-stranded and double-strandedform. Typical vectors useful in site-directed mutagenesis includevectors such as the M13 phage, for example, as disclosed by Messing etal., Third Cleveland Symposium on Macromolecules and Recombinant DNA,Editor A. Walton, Elsevier, Amsterdam (1981). These phage are readiblycommercially available and their use is generally well known to thoseskilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Vieira et al., Meth.Enzymol. 153:3 (1987)) can be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase 1Klenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells and clones are selected that include recombinant vectors bearingthe mutated sequence arrangement. After such a clone is selected, themutated protein region can be removed and placed in an appropriatevector for protein production, generally an expression vector of thetype that can be employed for transformation of an appropriate host.

Some deletions and insertions, and substitutions are not expected toproduct radical changes in the characteristics of EE proteins. However,when it is difficult to predict the exact effect of the substitution,deletion, or insertion in advance of doing so, one skilled in the artwill appreciate that the effect will be evaluated by routine screeningassays. For example, a variant typically is made by site-specificmutagenesis of the native EE OMP-encoding nucleic acid, expression ofthe variant nucleic acid in recombinant cell culture, and, optionally,purification from the cell culture, for example, by immunoaffinityadsorption on a column (to absorb the variant by binding it to at leastone remaining immune epitope). The activity of the cell lysate orpurified EE OMP molecule variant is then screened in a suitablescreening assay for the desired characteristic. For example, a change inthe immunological character of the OMP molecule, such as affinity for agiven antibody, is measured by a competitive type immunoassay. Changesin immunomodulation activity are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan.

A “fragment” is an immunoreactive fragment of an EE protein that has alength of from about 6 amoni acids to less than the full length EEprotein and includes a sequence that contains at least 6 consecutiveamino acids of a sequence of the EE protein. These fragments arecollectively referred to herein as “EE peptides.” In some embodiments,the fragment has at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 consecutiveamino acids of an EE protein sequence. The fragment can have a length ofat most, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, or 300 amino acids. In some embodiments, animmunoreactive fragment has from six to sixty amino acids, from six tofifty amino acids, from ten to fifty amino acids, from six to twentyamino acids, from eight to twenty amino acids, from ten to twenty aminoacids, from twelve to twenty amino acids or from twelve to seventeenamino acids. The immunoreactive fragments contemplated hereinspecifically exclude any fragment encoded by a 505 bp that is fromnucleotide 16,918 to 17,422 of the omp-1 gene cluster sequence, SEQ IDNO: 1, previously reported as the E. ewingii p-28-1 peptide. Thus, theimmunoreactive fragments as described herein specifically exclude any ofthe previously reported sequences for the 505-bp E. ewingii p28-1sequence, GenBank accession numbers: AF287961 (SEQ ID NOS 127-128),AF287962 (SEQ ID NOS 129-130), AF287963 (SEQ ID NOS 131-132), AF287964(SEQ ID NOS 133-134, AF287966 (SEQ ID NOS 135-136); or any fragmentthereof.

In some embodiments, the immunoreactive peptides are from six (6) aminoacids up to less than the full length EE protein, and are antigenic,i.e. are recognized by mammalian immune systems effectively. For thispurpose, the peptides comprise segments that are bacterial surfaceexposed, rather than bacterial cytoplasmic side-exposed or embeddedwithin the lipid bilayer membrane. Such surface exposed regions of EEproteins can be identified using computer programs using algorithms thatcan predict the three dimensional structure of the EE proteins based onthe hydrophobicity/hydrophilicity of the amino acid regions and therepeated β sheet model.

In some embodiments, the fragments comprise a sequence that is identicalto at least 6 consecutive amino acids of one or more of the variableloops depicted in Tables 2-5. In other0 embodiments, the fragment has asequence that comprises at least 6 consecutive amino acids in anycombination of one or more of the variable loops depicted in Tables 2-5.For example, the fragment can have a sequence from loops 1 and 2, loops1 and 3-1, loops 1 and 3-2, loops 1 and 4, loops 2 and 3-1, loops 2 and3-2, loops 2 and 4, loops 3-1 and 4, loops 3-2 and 4, loops 1 and 2 and3-1, loops 1 and 2 and 3-2, loops 2 and 3-1 and 4, loops 2 and 3-2 and4, loops 1 and 3-1 and 4, loops 1 and 3-2 and 4, loops 1 and 2 and 3-1and 4, or loops 1 and 2 and 3-2 and 4, etc.

In some embodiments, the immunoreactive fragments (or EE peptides)include the sequences set forth in Table 6. Thus, in one embodiment, theOMP-1-1 peptide includes the sequence, SEQ ID NO: 25; the OMP-1-2peptide includes the sequence, SEQ ID NO: 26; the OMP-1-3 peptideincludes the sequence, SEQ ID NO: 27; the OMP-1-4 peptide includes thesequence, SEQ ID NO: 28; the OMP-1-5 peptide includes the sequence, SEQID NO: 29; the OMP-1-6 peptide includes the sequence, SEQ ID NO: 30; theOMP-1-7 peptide includes the sequence, SEQ ID NO: 31; the OMP-1-8peptide includes the sequence, SEQ ID NO: 32; the OMP-1-9 peptideincludes the sequence, SEQ ID NO: 33; the OMP-1-10 peptide includes thesequence, SEQ ID NO: 34; the OMP-1-11 peptide includes the sequence, SEQID NO: 35; the OMP-1-12 peptide includes the sequence, SEQ ID NO: 36;the OMP-1-13 peptide includes the sequence, SEQ ID NO: 37; the OMP-1-14peptide includes the sequence, SEQ ID NO: 38; the OMP-1-15 peptideincludes the sequence, SEQ ID NO: 39; the OMP-1-16 peptide includes thesequence, SEQ ID NO: 40; the OMP-1-17 peptide includes the sequence, SEQID NO: 41; the OMP-1-18 peptide includes the sequence, SEQ ID NO: 42;and the OMP-1-19 peptide includes the sequence, SEQ ID NO: 43.

TABLE 6 E. ewingii OMP-1 peptide sequences used in ELISA (see Example 1)Peptide length SEQ (amino acid position ID NO: OMP-1 IDAmino acids sequence in the protein sequence) 25 OMP-1-1SCTEQEMKPAQQNGSSK 17 a.a (151-167) 26 OMP-1-2 EQHFALASELDTNGNQ 16 a.a(130-145) 27 OMP-1-3 VSAPSGYDDNIYAYFSI 17 a.a (123-139) 28 OMP-1-4FAIPRDTFFNNSIPY 15 a.a (144-150) 29 OMP-1-5 NSSSLISSNNHYTQLY 16 a.a(129-144) 30 OMP-1-6 KDNNQVQPKAHDSSSTD 17 a.a (148-164) 31 OMP-1-7KDPKDCSVKDAFRHL 15 a.a (130-144) 32 OMP-1-8 TEDKYLTSEQEVNDY 15 a.a(120-134) 33 OMP-1-9 ICKENDKPTPKEKKY 15 a.a (145-159) 34 OMP-1-10YRYFAIAREMNSSSNNQ 17 a.a (137-153) 35 OMP-1-11 KNSGHSSIDAHR 12 a.a(136-147) 36 OMP-1-12 IESDQNKFQPKNANSNS 17 a.a (154-170) 37 OMP-1-13SESSKEPQPKNPNSAGN 17 a.a (159-175) 38 OMP-1-14 KYYGLFREGTPQEEEH 16 a.a(146-161) 39 OMP-1-15 SRKDNANIGTTPQDKK 16 a.a (141-156) 40 OMP-1-16KIEDNQVQNKFTISNY 16 a.a (76-91) 41 OMP-1-17 QFYREGSNNYKF 12 a.a(123-134) 42 OMP-1-18 VQDTKSHIVDDNYR 14 a.a (121-144) 43 OMP-1-19SKQDNLNSDYVTLIN 15 a.a (171-185)

Also provided herein are fusion proteins in which a tag or one or moreamino acids from a heterologous protein are added to the amino orcarboxy terminus of the amino acid sequence of an EE protein or afunctional derivative thereof. At least one of the proteins or peptidescan be in a multimeric form. As used herein, the term “heterologousprotein” means a protein derived from a source other than the E. ewingiiomp-1 gene, operationally linked to a E. ewingii protein or a functionalderivative thereof, as disclosed in the present specification, to form achimeric or fusion E. ewingii protein or peptide. Typically, suchadditions are made to stabilize the resulting fusion protein or tosimplify purification of an expressed recombinant form of thecorresponding EE protein, variant, or peptide. Such tags are known inthe art. Representative examples of such tags include sequences whichencode a series of histidine residues, the Herpes simplex glycoproteinD, or glutathione S-transferase. Such a chimeric or fusion protein canhave a variety of lengths including, but not limited to, a length of atmost 100 residues, at most 200 residues, at most 300 residues, at most400 residues, at most 500 residues, at most 800 residues or at most 1000residues. Non-limiting examples of chimeric E. ewingii proteins includefusions of E. ewingii OMPs, or variants, or peptides: with immunogenicpolypeptides, such as flagellin and cholera enterotoxin; withimmunomodulatory polypeptides, such as IL-2 and B7-1; with tolerogenicpolypeptides; with another E. ewingii OMP, or variant, or peptide; andwith synthetic sequences. Other examples include linking the EE protein,or variant or peptide with an indicator reagent, an amino acid spacer,an amino acid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand or a combination ofthereof. The fusion proteins can have similar or substantially similarimmunoreactivity to EE antibodies as the EE proteins from which theyderive.

The EE polypeptides of the present invention can be used in a variety ofprocedures and methods, such as for the generation of antibodies,immunogenic composition and vaccines; for use in identifyingpharmaceutical compositions; for studying DNA/protein interaction; aswell as for diagnostic and screening methods.

Also provided are compositions of matter comprising one or more EEproteins, their functional derivatives and/or EE fusion proteins. Theisolated or purified polypeptide in such compositions can be in amultimeric form and can further include a carrier. The purifiedpolypeptide can be linked to an indicator reagent, an amino acid spacer,an amino acid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand, or a combination ofthese. Alternatively, one or more EE proteins or peptides may be linkedtogether.

Isolated Polynucleotides Coding for EE Polypeptides

The present invention also provides isolated polynucleotides as follows:

(a) a polynucleotide sequence encoding an immature EE protein OMP-1-1,OMP-1-2, OMP-1-3, OMP-1-4, OMP-1-5, OMP-1-6, OMP-1-7, OMP-1-8, OMP-1-9,OMP-1-10, OMP-1-11, OMP-1-12, OMP-1-13, OMP-1-14, OMP-1-15, OMP-1-16,OMP-1-17, OMP-1-18, or OMP-1-19, as described above;

(b) a polynucleotide sequence encoding a mature EE protein OMP-1-1,OMP-1-2, OMP-1-3, OMP-1-4, OMP-1-5, OMP-1-6, OMP-1-7, OMP-1-8, OMP-1-9,OMP-1-10, OMP-1-11 OMP-1-12, OMP-1-13, OMP-1-14, OMP-1-15, OMP-1-16,OMP-1-17, OMP-1-18, or OMP-1-19, as described above;

(c) a polynucleotide sequence encoding a functional derivative of an EEprotein of (a) or (b) above;

(d) a polynucleotide sequence encoding a fusion protein of an EE proteinor functional derivative of an EE protein;

(e) a polynucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c) or (d); and

(f) a polynucleotide sequence that hybridizes under highly stringenthybridization conditions with any of the nucleotide sequences in (a),(b), (c), (d) or (e).

The EE protein encoded by the polynucleotide of the invention includes:(1) a mature OMP-1-1 protein encoded by nucleotide 672-1484 of SEQ IDNO: 1; (2) a mature OMP-1-2 protein encoded by nucleotide 2116-2871 ofSEQ ID NO: 1; (3) a mature OMP-1-3 protein encoded by nucleotide3610-4395 of SEQ ID NO: 1; (4) a mature OMP-1-4 protein encoded bynucleotide 4486-5286 of SEQ ID NO: 1; (5) a mature OMP-1-5 proteinencoded by nucleotide 5380-6129 of SEQ ID NO: 1; (6) a mature OMP-1-6protein encoded by nucleotide 6216-7040 of SEQ ID NO: 1; (7) a matureOMP-1-7 protein encoded by nucleotide 7145-7921 of SEQ ID NO: 1; (8) amature OMP-1-8 protein encoded by nucleotide 8032-8679 of SEQ ID NO: 1;(9) a mature OMP-1-9 protein encoded by nucleotide 8772-9536 of SEQ IDNO: 1; (10) a mature OMP-1-10 protein encoded by nucleotide 9620-10387of SEQ ID NO: 1; (11) a mature OMP-1-11 protein encoded by nucleotide10477-11268 of SEQ ID NO: 1; (12) a mature OMP-1-12 protein encoded bynucleotide 11370-12188 of SEQ ID NO: 1; (13) a mature OMP-1-13 proteinencoded by nucleotide 12292-13113 of SEQ ID NO: 1; (14) a matureOMP-1-14 protein encoded by nucleotide 14530-15312 of SEQ ID NO: 1; (15)a mature OMP-1-15 protein encoded by nucleotide 15689-16450 of SEQ IDNO: 1; (16) a mature OMP-1-16 protein encoded by nucleotide 16861-17634of SEQ ID NO: 1; (17) a mature OMP-1-17 protein encoded by nucleotide18479-19222 of SEQ ID NO: 1; (18) a mature OMP-1-18 protein encoded bynucleotide 19558-20310 of SEQ ID NO: 1; and (19) a mature OMP-1-19protein encoded by nucleotide 21188-21967 of SEQ ID NO: 1.

The functional derivative encoded by the polynucleotide should not havethe sequence SEQ ID NO: 128, 130,132, 134, 136; or any fragment thereof;and each functional derivative should have a specific binding affinityfor an anti-E. ewingii antibody.

In some embodiments, the functional derivative encoded by thepolynucleotide comprises a sequence which is at least 85%, 90%, 95% or98% identical to the sequence of the mature OMP-1 protein (1)-(19), asdescribed above.

In some embodiments, the functional derivative encoded by thepolynucleotide comprises an immunoreactive fragment that has a length offrom 6 amino acids to less than the full length of the EE protein andcomprises 6 or more consecutive amino acids from the followingsequences: (1) SEQ ID NO: 137-155; (2) SEQ ID NO: 156-173; (3) SEQ IDNO: 174-191; (4) SEQ ID NO: 192-208; (5) SEQ ID NO: 209-227; or (6) anycombination of the sequences (1)-(5); wherein each immunoreactivefragment has a specific binding affinity for an anti-E. ewingiiantibody.

In other embodiments, the EE protein encoded by the polynucleotidecomprises a sequence selected from the group consisting of: (1) SEQ IDNO: 3 corresponding to an immature OMP-1-1 protein; (2) SEQ ID NO: 4corresponding to an immature OMP-1-2 protein; (3) SEQ ID NO: 6corresponding to an immature OMP-1-3 protein; (4) SEQ ID NO: 7corresponding to an immature OMP-1-4 protein; (5) SEQ ID NO: 8corresponding to an immature OMP-1-5 protein; (6) SEQ ID NO: 9corresponding to an immature OMP-1-6 protein; (7) SEQ ID NO: 10corresponding to an immature OMP-1-7 protein; (8) SEQ ID NO: 11corresponding to an immature OMP-1-8 protein; (9) SEQ ID NO: 12corresponding to an immature OMP-1-9 protein; (10) SEQ ID NO: 13corresponding to an immature OMP-1-10 protein; (11) SEQ ID NO: 14corresponding to an immature OMP-1-11 protein; (12) SEQ ID NO: 15corresponding to an immature OMP-1-12 protein; (13) SEQ ID NO: 16corresponding to an immature OMP-1-13 protein; (14) SEQ ID NO: 17corresponding to an immature OMP-1-14 protein; (15) SEQ ID NO: 18corresponding to an immature OMP-1-15 protein; (16) SEQ ID NO: 19corresponding to an immature OMP-1-16; (17) SEQ ID NO: 20 correspondingto an immature OMP-1-17 protein; (18) SEQ ID NO: 21 corresponding to animmature OMP-1-18 protein; (19) SEQ ID NO: 22 corresponding to animmature OMP-1-19 protein.

In one embodiment, the polynucleotide comprises a portion of anucleotide sequence SEQ ID NO: 1.

It is understood that the polynucleotides encoding the EE polypeptidescan have a different sequence than the nucleotide sequence shown inTable 1 due to the degeneracy of the genetic code. Thus, also includedwithin the scope of this invention are the functional equivalents of theherein-described isolated polynucleotides and derivatives thereof. Forexample, the nucleic acid sequences depicted in SEQ ID NO: 1 can bealtered by substitutions, additions or deletions that provide forfunctionally equivalent molecules. Due to the degenracy of nucleotidecoding sequences, other DNA sequences which encode substantially thesame amino acid sequences as depicted in Tables 1-6 can be used in thepractice of the present invention. These include but are not limited tonucleotide sequences comprising all or portions of SEQ ID NO: 1 thatencode for EE polypeptides, which are altered by the substitution ofdifferent codons that encode a Functionally equivalent amino acidresidue within the sequence.

In addition, the polynucleotide can comprise a nucleotide sequence whichresults from a addition, deletion or substitution of at least onenucleotide to the 5′-end and/or the 3′-end of the nucleic acid segmentsof SEQ ID NO: 1, or a derivative thereof. Any polynucleotide can be usedin this regard, provided that its addition, deletion or substitutiondoes not substantially alter the amino acid sequence of the EE protein,or functional derivatives or fusion proteins thereof, encoded by thepolynucleotide sequence. Moreover, the polynucleotide of the presentinvention can, as necessary, have restriction endonuclease recognitionsites added to its 5′-end and/or 3′-end. All variations of thenucleotide sequence of the EE omp-1 gene and fragments thereof permittedby the genetic code are, therefore, included in this invention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

Finally, the isolated polynucleotides of the invention expressly excludeany polynucleotide that encodes for a peptide that is the 505-bp E.ewingii p28-1 peptide deposited in GenBank accession numbers: AF287961(SEQ ID NOS 127-128), AF287962 (SEQ ID NOS 129-130), AF287962 (SEQ IDNOS 131-132), AF287964 (SEQ ID NOS 133-134, AF287966 (SEQ ID NOS135-136); or any fragment thereof.

Isolation of Polynucleotides

The isolated EE polynucleotides coding for EE polypeptides can beisolated from a biological sample (e.g. of mammalian or tick origin)containing EE RNA or DNA.

The polynucleotide can be isolated from a biological sample containingEE RNA using the techniques of cDNA cloning and subtractivehybridization. The polynucleotide can also be isolated from a cDNAlibrary using a homologous probe, i.e., a probe comprising sequencessubstantially identical or complementary to portions or all of thepolynucleotide sequence SEQ ID NO: 1; or with antibodies immunospecificfor an EE OMP protein or peptide to identify clones containing suchpolynucleotides.

The polynucleotide can be isolated from a biological sample containinggenomic DNA or from a genomic library. Suitable biological samplesinclude, but are not limited to, whole organisms, organs, tissues, bloodand cells. The method of obtaining the biological sample will varydepending upon the nature of the sample.

One skilled in the art will realize that EE polynucleotides may also beisolated from a number of infected eukaryotes (for example, mammals,birds, fish and humans) that may contain the EE protein genes.

Synthesis of Polynucleotides

Isolated polynucleotides of the present invention include thosechemically synthesized. For example, a polynucleotide with thenucleotide sequence which codes for the expression product of an EEprotein gene can be designed and, if necessary, divided into appropriatesmaller fragments. Then an oligomer which corresponds to thepolynucleotide, or to each of the divided fragments, can be synthesized.Such synthetic oligonucleotides can be prepared, for example, by thetriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185-3191(1981) or by using an automated DNA synthesizer.

An oligonucleotide can be derived synthetically or by cloning. Ifnecessary, the 5′-ends of the oligomers can be phosphorylated using T4polynucleotide kinase. Kinasing of single strands prior to annealing orfor labeling can be achieved using an excess of the enzyme. If kinasingis for the labeling of probe, the ATP can contain high specific activityradioisotopes. Then, the DNA oligomer can be subjected to annealing andligation with T4 ligase or the like.

Also encompassed by the present invention, are single strandedpolynucleotides, hereinafter referred to as antisense polynucleotides,having sequences which are complementary to the DNA and RNA sequenceswhich encode the EE proteins and peptides, or their functional variants.

DNA Constructs Comprising a Polynucleotide Encoding an EE Polypeptide,and Cells Containing These Constructs

The EE polynucleotides described herein are useful for producing the EEpolypeptides. For example, an RNA molecule encoding an EE polypeptidecan be used in a cell-free translation system to prepare an isolatedpolypeptide corresponding to an EE protein, its functional derivativesor fusion proteins. Alternatively, a DNA molecule encoding an EEpolypeptide can be introduced into an expression vector and used totransform cells.

Accordingly, in another aspect, the present invention relates to anrecombinant DNA molecule comprising, 5′ to 3′, a promoter effective toinitiate transcription in a host cell and one or more of theabove-described EE polynucleotides. In another embodiment, the presentinvention relates to a recombinant DNA molecule comprising a vector andan above-described polynucleotide.

In another embodiment, the present invention relates to a nucleic acidmolecule comprising a transcriptional control region functional in acell, a sequence complimentary to an RNA sequence encoding an amino acidsequence corresponding to an above-described EE polypeptide, and atranscriptional termination region functional in the cell.

In one embodiment, the above-described molecules are isolated and/orpurified DNA molecules.

In another embodiment, the present invention relates to a cell ornon-human organism that has been altered to express the EE polypeptidesand so contains one or more of the above-described nucleic acidmolecules.

In another embodiment, the polypeptide is purified from cells which havebeen altered to express the polypeptide.

As used herein, a cell, or organism, is said to be “altered to express adesired polypeptide” when the cell, or organism, through geneticmanipulation, is made to produce a protein which it normally does notproduce or which it normally produces at low levels. One skilled in theart can readily adapt procedures for introducing and expressing eithergenomic, cDNA, or synthetic sequences into either eukaryotic orprokaryotic cells.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression can vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3′ to the EE polypeptide codingsequence can be obtained by the above-described methods. This region canbe retained for its transcriptional termination regulatory sequences,such as termination and polyadenylation. Thus, by retaining the3′-region naturally contiguous to the DNA sequence encoding an EEprotein gene, the transcriptional termination signals can be provided.Where the transcriptional termination signals are not satisfactorilyfunctional in the expression host cell, then a 3′ region functional inthe host cell can be substituted.

Two DNA sequences (such as a promoter region sequence and an EEpolypeptide coding sequence) are said to be operably linked if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region sequence to direct the transcription ofan EE polypeptide coding sequence, or (3) interfere with the ability ofthe EE polypeptide coding sequence to be transcribed by the promoterregion sequence. Thus, a promoter region would be operably linked to aDNA sequence if the promoter were capable of effecting transcription ofthat DNA sequence.

The present invention encompasses the expression of the EE polypeptidecoding sequence in either prokaryotic or eukaryotic cells. Prokaryotichosts are, generally, the most efficient and convenient for theproduction of recombinant proteins and, therefore, are suitable for theexpression of the EE polypeptide coding sequence.

Prokaryotes most frequently are represented by various strains of E.coli. However, other microbial strains can also be used, including otherbacterial strains. In prokaryotic systems, plasmid vectors that containreplication sites and control sequences derived from a speciescompatible with the host can be used. Examples of suitable plasmidvectors include, but are not limited to, pBR322, pUC18, pUC19, pUC118,pUC119 and the like; suitable phage or bacteriphage vectors includeλgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo,pKRC and the like. In some examples, the selected vector of the presentinvention has the capacity to replicate in the selected host cell.

Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However,under such conditions, the peptide will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

To express EE polypeptides in a prokaryotic cells, it is necessary tooperably link the EE protein coding sequence to a functional prokaryoticpromoter. Such promoters can be either constitutive or regulatable(i.e., inducible or derepressible). Examples of constitutive promotersinclude, but are not limited to, the int promoter of bacteriophage λ,the bla promoter of the β-lactamase gene sequence of pBR322, and the CATpromoter of the chloramphenicol acetyl transferase gene sequence ofpBR325, and the like. Examples of inducible prokaryotic promotersinclude, but are not limited to, the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ, lacI, and gaIpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)) and the ξ-28-specific promoters of B. subtilis(Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of thebacteriopages of Bacillus (Gryezan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryoticpromoters are reviewed by Glick (J. Ind. Microbiol. 1:277-282 (1987));Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev.Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell may also require the presence ofa ribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et al(Ann. Rev. Microbiol. 35:365-404 (1981)).

The selection of control sequences, expression vectors, transformationmethods, and the like, are dependent on the type of host cell to expressthe gene. As used herein, “cell”, “cell line”, and “cell culture” can beused interchangeably and all such designations include progeny. Thus,the words “transformants” or “transformed cells” include the primarysubject cell and cultures derived therefrom, without regard to thenumber of transfers. It is also understood that all progeny can not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. However, as defined, mutant progeny have the samefunctionality as that of the originally transformed cell. Host cellswhich can be used in the expression systems of the present invention arenot strictly limited, provided that they are suitable for use in theexpression of the EE polypeptides of interest. Suitable hosts includeeukaryotic cells.

Some examples of suitable eukaryotic hosts include, but are not limitedto, yeast, fungi, insect cells, mammalian cells either in vivo, or intissue culture. Suitable mammalian cells include HeLa cells, cells offibroblast origin such as VERO or CHO-K1, or cells of lymphoid originand their derivatives.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences.

Another type of host is an insect cell, for example Drosophila larvae.Using insect cells as hosts, the Drosophila alcohol dehydrogenasepromoter can be used, Rubin, Science 240:1453-1459 (1988).Alternatively, baculovirus vectors can be engineered to express largeamounts of EE polypeptides in insect cells (Jasny, Science 238:1653(1987); Miller et al., In: Genetic Engineering (1986), Setlow, J. K., etal., eds., Plenum, Vol. 8, pp. 277-297).

Different host cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, cleavage) of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the desired modification and processingof the foreign protein expressed.

Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes. Theseenzymes are produced in large quantities when yeast are grown in mediumsrich in glucose. Known glycolytic gene sequences can also provide veryefficient transcriptional control signals.

Yeast can provide substantial advantages in that it can also carry outpost-translational peptide modifications. A number of recombinant DNAstrategies exist which utilize strong promoter sequences and high copynumber of plasmids which can be utilized for production of the desiredproteins in yeast. Yeast recognizes leader sequences on cloned mammaliangene sequence products and secretes peptides bearing leader sequences(i.e., pre-peptides). For a mammalian host, several possible vectorsystems are available for the expression of EE polypeptides.

A wide variety of transcriptional and translational regulatory sequencescan be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals can be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, can be employed. Transcriptionalinitiation regulatory signals can be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

As discussed above, expression of EE polypeptides in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Examples of eukaryotic promoters include, but are notlimited to, the promoter of the mouse metallothionein 1 gene sequence(Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter ofHerpes virus (McKnight, Cell 31:355-365 (1982)), the SV40 early promoter(Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA)81:5951-5955 (1984)) and the CMV immediate-early gene promoter (Thomsenet al., Proc. Natl. Acad. Sci (USA) 81:659-663 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it may bedesirable to ensure that the linkage between a eukaryotic promoter andan EE polypeptide coding sequence does not contain any interveningcodons which are capable of encoding a methionine (i.e., AUG). Thepresence of such codons results either in a formation of a fusionprotein (if the AUG codon is in the same reading frame as the EEpolypeptide coding sequence) or a frame-shift mutation (if the AUG codonis not in the same reading frame as the EE polypeptide coding sequence).

A nucleic acid molecule encoding an EE polypeptide and an operablylinked promoter can be introduced into a recipient prokaryotic oreukaryotic cell either as a non-replicating DNA (or RNA) molecule, whichcan either be a linear molecule or a closed covalent circular molecule.Since such molecules are incapable of autonomous replication, theexpression of the gene can occur through the transient expression of theintroduced sequence. Alternatively, permanent expression can occurthrough the intergration of the introduced DNA sequence into the hostchromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markercan provide for prototrophy to an auxotrophic host, biocide resistance,e.g., antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements can also be needed for optimalsynthesis of single chain binding protein mRNA. These elements caninclude splice signals, as well as transcription promoters, enhancersignal sequences, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, Molec.Cell. Biol. 3:280 (1983).

In some embodiments, the introduced nucleic acid molecule will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors canbe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector can be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

Examples of prokaryotic vectors include, but are not limited to, plasmidsuch as those capable of replication in E. coli (such as, for example,pBR322, ColE1, pSC101, pACYC 184, πVX. Such plasmids are, for example,disclosed by Sambrook (Molecular Cloning: A Labroatory Manual, secondedition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring HarborLaboratory, 1989), Bacillus plasmids include pC194, pC221, pT127, andthe like. Such plasmids are disclosed by Gryezan (In: The MolecularBiology of the Bacilli, Academic Press, NY (1982), pp. 307-329).Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J.Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages suchas φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akaderniai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al (Rev. Infect.Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

Examples of suitable eukaryotic plasmids include, but are not limitedto, BPV, vaccinia, SV40, 2-micron circle, and the like, or theirderivatives. Such plasmids are well known in the art (Botstein et al.,Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach,Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10:39-48(1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol 3,Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) can be introducedinto an appropriate host cell by any of a variety of suitable means,e.g., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofEE polypeptide. This can take place in the transformed cells as such, orfollowing the induction of these cells to differentiate (for example, byadministration of bromodeoxyuracil to neuroblastoma cells or the like).

Nucleic Acid Probes and Primers for the Specific Detection of E. Ewingii

The EE polynucleotides described herein are also useful for designinghybridization probes for isolating and identifying cDNA clones andgenomic clones encoding the EE proteins, peptides or allelic formsthereof. Such hybridization techniques are known to those of skill inthe art.

Therefore, in another embodiment, a nucleic acid probe is provided forthe specific detection of the presence of one or more EE polynucleotidesin a sample comprising the above-described isolated polynucleotides orat least a fragment thereof, which binds under stringent conditions, orhighly stringent conditions, to EE polynucleotides.

The term “stringent conditions” as used herein is the binding whichoccurs within a range from about Tm 5° C. (5° C. below the meltingtemperature Tm of the probe) to about 20° C. to 25° C. below Tm. Theterm “highly stringent hybridization conditions” as used herein refersto conditions of: at least about 6×SSC and 1% SDS at 65° C., with afirst wash for 10 minutes at about 42° C. with about 20% (v/v) formamidein 0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65°C.

In one embodiment, the isolated nucleic acid probe consisting of 10 to1000 nucleotides (for example: 10 to 500, 10 to 250, 10 to 100, 10 to50, 10 to 35, 20 to 1000, 20 to 500, 20 to 250, 20 to 100, 20 to 50, or20 to 35, etc.) which hybridizes preferentially to RNA or DNA of EE butnot to RNA or DNA of non-EE organisms, wherein said nucleic acid probeis or is complementary to a nucleotide sequence consisting of at least10 consecutive nucleotides, or 15, 20, 25, 30, 50, 100, 250, 500, 600,700, 800, or 900 consecutive nucleotides, or along the entire length, ofone or more of the EE polynucleotides described above.

In some embodiments, the nucleic acid probe comprises a polynucleotidesequence encoding a polypeptide that corresponds to one or more of thevariable loop sequences in Tables 2-6. Such probes would hybridize witha specific polynucleotide encoding a polypeptide corresponding to avariable sequence in each EE OMP protein and so will be specific to EE,as opposed to the other ehrlichia species. Methods for designing probesthat are specific for EE polynucleotide sequences based on the sequencealignment provided in FIG. 7 are well known in the art.

In other embodiments, the nucleic acid probe can be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain another nucleic acid molecule of the present invention. Achromosomal DNA or cDNA library can be prepared from appropriate cellsaccording to recognized methods in the art (see e.g. Molecular Cloning:A Laboratory Manual, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989).

Such hybridization probes can have a sequence which is at least 90%,95%, 98%, 99% or 100% complementary with a sequence contained within thesense strand of a DNA molecule which encodes each of the EE proteins orwith a sequence contained within its corresponding antisense strand.Such hybridization probes bind to the sense or antisense strand understringent, or highly stringent, conditions.

The term “stringent conditions” as used herein is the binding whichoccurs within a range from about Tm 5° C. (5° C. below the meltingtemperature Tm of the probe) to about 20° C. to 25° C. below Tm. Theterm “highly stringent hybridization conditions” as used herein refersto conditions of: at least about 6×SSC and 1% SDS at 65° C., with afirst wash for 10 minutes at about 42° C. with about 20% (v/v) formamidein 0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65°C.

The probes can be used in a number of assays, such as Northern assays todetect transcripts of EE omp-1 homologous genes and in Southern assaysto detect EE omp-1 homologous genes. In some examples, the identity ofprobes which are e.g. 200 nucleotides in length and have fullcomplementarity with a portion of the antisense strand of adouble-stranded DNA molecule which encodes the EE proteins can bedetermined using EE protein-encoding segments of the nucleotide sequenceSEQ ID NO: 1.

The hybridization probes of the present invention can be labeled bystandard labeling techniques such as with a radiolabel, enzyme label,fluorescent label, biotin-avidin label, chemiluminescence, and the like.After hybridization, the probes can be visualized using known methods.

The nucleic acid probes of the present invention include RNA, as well asDNA probes, such probes being generated using techniques known in theart.

In one embodiment of the above described method, a nucleic acid probe isimmobilized on a solid support. Examples of such solid supports include,but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art.

The EE polynucleotides disclosed herein are also useful for designingprimers for polymerase chain reaction (PCR), a technique useful forobtaining large quantities for cDNA molecules that encode the EEpolypeptides. PCR primers can also be used for diagnostic purposes.

Thus, also included are oligonucleotides that are used as primers inpolymerase chain reaction (PCR) technologies to amplify transcripts ofthe genes which encode the EE polypeptides, or portions of suchtranscripts. In some examples, the primers comprise a minimum of about12 to 15 nucleotides and a maximum of about 30 to 35 nucleotides. Theprimers can have a G+C content of 40% or greater. Such oligonucleotidesare at least 98% complementary with a portion of the DNA strand, i.e.,the sense strand, which encodes the EE protein, or a portion of itscorresponding antisense strand. In some embodiments, the primer has atleast 99% complementarity, or 100% complementarity, with such sensestrand or its corresponding antisense strand. Primers which have 100%complementarity with the antisense strand of a double-stranded DNAmolecule encoding an EE protein have a sequence which is identical to asequence contained within the sense strand.

One skilled in the art can readily design such probes based on thesequences disclosed herein using methods of computer alignment andsequence analysis known in the art (see, for example, Molecular Cloning:A Laboratory Manual, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989). For example, theidentity of primers which are 15 nucleotides in length and have fullcomplementarity with a portion of the antisense strand of adouble-stranded DNA molecule which encodes the EE proteins can bedetermined using the EE protein segments of the nucleotide sequence, SEQID NO: 1.

In some embodiments, the primers have one or more of the followingfeatures: (i) the 3′ end of the primer set is either C or G; (ii) atleast the 3′ end of each primer has the same sequence as the target DNA(or its reverse complement), i.e., the 5′ end can have variations andstill produce a proper PCR product; (iii) the primer is about 20 bplong; (iv) both members of a primer set are around the same length; (v)the GC total and AT total for both primers in a set are the same, orvery similar; (vi) the primer should be designed after performing aBLAST search on each primer to determine if there could be alignment foramplification of an unwanted species; and/or (vii) use the formula4×(G+C)+2×(A+T)=PCR Annealing Temperature (±5° C.) for a primer set. Forexample, Ohashi N, et al. (2001) Infection and Immunity, 69:2083-91, theentire contents of which are incorporated herein by reference, describesmultiple primers designed to detect all 22 p30 proteins of E. canis. Asimilar strategy can be used for E. ewingii primer design.

In one embodiment, the primers are designed to amplify a target DNAsegment that is useful for diagnostic purposes. The target sequence canhave a length of 50-300 nucleotide bases, 50-200 bases, 80-200 bases,100-300, 300-600, 600-800, 600-1000, or 800-1000 bases. In someembodiments, the target DNA comprises a polynucleotide sequence encodinga polypeptide comprising one or more of the variable loop sequences inTables 2-6. Such primers would amplify a specific polynucleotide segmentencoding a polypeptide whose sequence corresponds to a variable sequencein each EE OMP protein and so will be specific to EE, as opposed toother ehrlichia species. Methods for designing primers that are specificfor EE polynucleotide sequences based on the sequence alignment providedin FIGS. 7 and 8 are well known in the art.

In another embodiments, the target DNA segment has a nucleotide sequencethat encodes a polypeptide that is conserved among E. ewingii strains,but is distinct from other Ehrlichia strains. Examples of conservedsequences among E. ewingii strains are shown as darkened areas in theattached sequence alignment (FIGS. 7 and 8). Using as target DNAsequences that encode for conserved regions of EE proteins increases thesensitivity of the PCR reaction because there are more than two copiesof the target sequence in the genome. This increases the PCR'ssensitivity more than two fold. For example, U.S. Pat. No. 6,432,649 toStich et al., the entire contents of which is incorporated herein byreference, describes methods of designing such primers based on sequencealignments of E. canis and E. chaffeensis for the specific diagnosis ofeach of these species. A similar method can be employed to designoptimal primer sets for E. ewingii diagnosis.

In other embodiments, the primers are designed for nested PCR, or targetmore than two regions of the target sequence to increase sensitivity.Nested PCR is a conventional PCR with a second round of amplificationusing a different set of primers. This second set of primers is specificto a sequence found within the target DNA of the initial conventionalPCR amplicon. The use of a second amplification step with the “nested”primer set results in a reduced background from products amplifiedduring the initial PCR due to the nested primers' additional specificityto the region. The amount of amplicon produced is increased as a resultof the second round of amplification and due to a reduction in anyinhibitor concentrations. For example, the first set of primers cantarget variable loops 1 and 4, and the second nested primer set cantarget variable loops 2 or 3.

Primer design choices that can increase the PCR reaction's specificityinclude using primers that border the target DNA sequence together withhigher annealing temperatures.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting an a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product complementary to a nucleic acid strand is induced. Theconditions include the presence of nucleotides and an inducing agentsuch as a DNA polymerase and a suitable temperature and pH. The primermay be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, source ofprimer and the method used. For example, for diagnostic applications,the oligonucleotide primer typically contains 15-30 or more nucleotidesdepending on the complexity of the target sequence. Primers with fewernucleotides may also be used.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementary with the sequence or hybridize therewith andthereby form the template for the synthesis of the extension product.

Antibodies

Also contemplated herein is an isolated or purified antibody havingspecific binding affinity to an EE polypeptide as described above.

An antibody that has a “specific binding affinity” to an EE polypeptide,is an antibody that binds with a substantially greater affinity to theEE polypeptide, than to an E. canis or E. chaffeensis protein.

Any of a variety of routine assays can be used for detectingantigen-antibody complexes, the presence of which is an indicator ofselective binding. Such assays include, without limitation,enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, westernblotting, enzyme immunoassays, fluorescence immunoassays, luminescentimmunoassays and the like. Methods for detecting a complex between apeptide and an antibody, and thereby identifying and antibody withspecific binding affinity to an EE polypeptide are well known to thoseskilled in the art and are described, for example, in ANTIBODIES: ALABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring HarborLaboratory Press, 2.sup.nd ed. 1998a); and USING ANTIBODIES: ALABORATORY MANUAL: PORTABLE PROTOCOL No. 1 (Edward Harlow & David Lane,Cold Spring Harbor Laboratory Press, 1998b), which are herebyincorporated by reference in their entirety.

In one embodiment, the “specific binding affinity” of an antibody isdefined as an ELISA assay result, where the ratio of E. chaffeensis orE. canis polypeptide reactivity/control plasma reactivity is ˜1.00, andwhere E. ewingii polypeptide reactivity yields an OD_(405nm)-OD_(492nm)value greater than the mean OD_(405nm)-OD_(492nm) of preinfectioncontrol plasma+three standard deviations. In this example, theantibodies can be obtained from a subject infected with E. ewingii. Thecontrol plasma can include preinfection plasma, plasma from subjectsinfected with anything other than E. ewingii, or plasma from subjectesinfected with E. chaffeensis or E. canis.

The EE polypeptides can be used to produce antibodies or hybridomas. Oneskilled in the art will recognize that if an antibody is desired, such apolypeptide would be generated as described herein and used as animmunogen.

The produced antibodies are useful research tools for diagnostic andscreening purposes, for identifying cells, such as granulocytes,infected with E. ewingii and for purifying the major outer membraneprotein of E. ewingii from partially purified preparations by affinitychromatography. Such antibodies are also useful for identifyingbacterial colonies, particularly colonies of genetically-engineeredbacteria, that are expressing the major outer membrane protein of E.ewingii.

The antibodies described herein can also be used in a composition to beadministered to a subject in need thereof, to reduce the level of E.ewingii infection in the subject. Such a reduction refers to a reductionor elimination of clinical signs and symptoms of E. ewingii infection inthe subject. Alternatively the antibody can be used to prevent infectionwith E. ewingii in a subject.

The antibodies include monoclonal and polyclonal antibodies, as well asfragments of these antibodies. The invention further includes singlechain antibodies. Antibody fragments which contain the idiotype of themolecule can be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragment; the Fab′fragments, Fab fragments, and Fv fragments.

In one embodiment, the antibodies to EE polypeptides are produced inhumans, or are “humanized” (i.e. non-immunogenic in a human) byrecombinant or other technology. Humanized antibodies can be produced,for example by replacing an immunogenic portion of an antibody with acorresponding, but non-immunogenic portion (i.e. chimeric antibodies)(Robinson, R. R. et al., International Patent PublicationPCT/US86/02269; Akira, K. et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison, S. L., etal., European Patent Application 173,494; Neuberger, M. S. et al., PCTApplication WO 86/01533; Cabilly, S. et al., European Patent Application125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A. Y. etal., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al.,J. Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad.Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Canc. Res. 47:999-1005(1987); Wood, C. R. et. al., Nature 314:446-449 (1985); Shaw et al., J.Natl. Cancer Inst. 80:1553-1559 (1988)). General reviews of “humanized”chimeric antibodies are provided by Morrison, S. L. (Science,229:1202-1207 (1985)) and by Oi, V. T. et al., BioTechniques 4:214(1986)). Suitable “humanized” antibodies can be alternatively producedby CDR or CEA substitution (Jones, P. T. et. al., Nature 321:552-525(1986); Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. etal., J. Immunol. 141:4053-4060 (1988)).

In another embodiment, the present invention relates to a hybridomawhich produces the above-described monoclonal antibody. A hybridoma isan immortalized cell line which is capable of secreting a specificmonoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam. The Netherlands(1984); St. Groth et al., J. Immunol. Methods 35:1-21 (1980)).

Any animal (mouse, rabbit, and the like) which is known to produceantibodies can be immunized with the selected polypeptide. Methods forimmunization are well known in the art. Such methods includesubcutaneous or interperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of polypeptide usedfor immunization will vary based on the animal which is immunized, theantigenicity of the polypeptide and the site of injection.

The polypeptide can be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, and allowed to become monoclonalantibody producing hybridoma cells. Any one of a number of methods wellknown in the art can be used to identify the hybridoma cell whichproduces an antibody with the desired characteristics. These includescreening the hybridomas with an ELISA assay, western blot analysis, orradioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124 (1988)).Hybridomas secreting the desired antibodies are cloned and the class andsubclass is determined using procedures known in the art (Campbell,Monoclonal Antibody Technology: Laboratory Techniques in Biochemistryand Molecular Biology, supra (1984)).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures.

In another embodiment, the above-described antibodies are detectablylabeled. Antibodies can be detectably labeled through the use ofradioisotopes, affinity labels (such as biotin, avidin, and the like),enzymatic labels (such as horseradish peroxidase, alkaline phosphatase,and the like) fluorescent labels (such as FITC or rhodamine, and thelike), paramagnetic atoms, and the like. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Stemberger etal., J. Histochem. Cytochem. 18:315 (1970); Bayer et al., Meth. Enzym.62:308 (1979); Engval et al., Immunol. 109:129 (1972); Goding, J.Immunol. Meth. 13:215 (1976)). The labeled antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays toidentify cells or tissues which express a specific polypeptide.

In another embodiment, the above-described antibodies are immobilized ona solid support. Examples of such solid supports include plastics suchas polycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10 (1986); Jacoby etal., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilizedantibodies can be used for in vitro, in vivo, and in situ assays as wellas in immunochromatography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific polypeptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides”, In SyntheticPeptides, A User's Guide, W. H. Freeman, NY, pp. 289-307 (1992), andKaspczak et al., Biochemistry 28:9230-8 (1989).

Anti-peptide peptides can be generated in one of two fashions. First theanti-peptide peptides can be generated by replacing the basic amino acidresidues found in the EE polypeptide sequence with acidic residues,while maintaining hydrophobic and uncharged polar groups. For example,lysine, arginine, and/or histidine residues are replaced with asparticacid or glutamic acid and glutamic acid residues are replaced by lysine,arginine or histidine.

Diagnostic Methods

Also contemplated herein are diagnostic methods that use the EEpolypeptides, EE polynucleotides, EE fusion proteins and EE antibodiesdescribed herein.

Samples used in the diagnostic methods are samples obtained from asubject that is suspected of having, or having has, E. ewingiiinfection. Subjects not infected with EE do not have EE DNA, mRNA,protein, or antibody.

The subject may be a human or any animal that can be infected with E.ewingii. Such subjects include, but should not be limited to, humans,horses, deer, cattle, pigs, sheep, dogs, cats and chicken.

The test sample may be a biological fluid such as serum, plasma, wholeblood, urine, or saliva, or may be tissue, cells, protein or membrane ornucleic acid extract of cells, obtained from a subject. The sample usedin the methods will vary based on the assay format, the detection methodand the nature of the tissues, cells or extracts to be assayed.

For example, in some embodiments, it is advantageous to use more thanone EE polypeptide as antigents in diagnostic methods. Since currentlyno E. ewingii-specific serodiagnosis is available, for serodiagnosis ofE. ewingii infection in either humans and animals, use of a combinationof EEOMP-1s (i.e. EE OMP-1 proteins or functional derivatives thereof)as the antigen can provide sensitive and specific serodiagnosis. Use ofmultiple EE polypeptides can provide more sensitive diagnosis than theuse of a single EE OMP-1 antigen. Not all humans and dogs developantibodies to every EEOMP-1 protein. Therefore, the use of a combinationof EE polypeptides (e.g. a combination of EE polypeptides correspondingto all OMP-1 proteins) as antigens provides a more comprehensivecoverage of antibody responses. Furthermore, the entire EEOMP-1 aminoacid sequences disclosed herein can help optimize peptide antigens toprovide desired specificity and sensitivity to detect potentiallydiverse E. ewingii strains in the field.

Diagnostic Methods Using EE Polypeptides and Antibodies

The present invention also provides a method for detecting the presenceof antibodies specific to E. ewingii in a test sample. The methodincludes contacting a test sample suspected of comprising antibodiesspecific for E. ewingii with one or more E. ewingii polypeptides, asdescribed herein, under conditions that allow polypeptide/antibodycomplexes to form; and assaying for the formation of a complex betweenantibodies in the test sample and the one or EE polypeptides.Accordingly, detecting the formation of such a complex is an indicationthat antibodies specific for E. ewingii are present in the test sample.

Another aspect provides for a method for detecting the presence of E.ewingii polypeptides in a test sample. The method includes contacting atest sample suspected of comprising E. ewingii polypeptides with one ormore E. ewingii antibodies that specifically bind to at least oneepitope of an E. ewingii OMP protein or peptide, as described herein,under conditions that allow polypeptide/antibody complexes to form; andassaying for the formation of a complex between polypeptides in the testsample and the one or EE antibodies. Accordingly, detecting theformation of such a complex is an indication that E. ewingiipolypeptides are present in the test sample.

The presence of EE polypeptides, or antibodies to EE polypeptides, mayindicate exposure to E. ewingii, the potential need for therapy of anaffected subject, or EE contamination of a biological sample.

For ease of detection, the isolated EE polypeptide or antibody can beattached to a substrate such as a column, plastic dish, matrix, ormembrane, such as nitrocellulose. The test sample may be untreated,subjected to precipitation, fractionation, separation, or purificationbefore combining with the isolated polypeptide. Conditions forincubating an EE polypeptide or antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay.

Interactions between antibodies and polypeptide can be detected in anumber of ways well know to those skilled in the art. These include, butare not limited to, radiometric, calorimetric, or fluorometric means,size-separation, or precipitation. These assays include, but are notlimited to, a microtiter plate assay, a reversible flow chromatographicbinding assay, an enzyme linked immunosorbent assay, a radioimmunoassay,a hemagglutination assay, a western blot assay, a fluorescencepolarization immunoassay, an indirect immunofluorescence assay,diffusion based Ouchterlony, or rocket immunofluorescent assays.Examples of such assays can be found in Chard, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).In one example, detection of the antibody-polypeptide complex is byaddition of a secondary antibody that is coupled to a detectable tag,such as for example, an enzyme, fluorophore, or chromophore. Formationof the complex is indicative of the presence of E. ewingii proteins, oranti-E. ewingii antibodies in the subject from whom the test sample wasobtained. Thus, the method is used to determine whether a subject isinfected with E. ewingii.

In some embodiments, the method employs an enzyme-linked immunosorbentassay (ELISA) or a Western immunoblot procedure. Such methods arerelatively simple to perform and do not require special equipment aslong as membrane strips are coated with a high quality antigen.Accordingly, it is possible to use a recombinant form of the EEpolypeptides since such proteins and peptides, typically, are more pureand consistent in quality than a their purified form.

Diagnostic Methods using EE Primers and Probes

The probes and primers described herein can be designed and useddiagnostically for determining whether a subject has been infected withan E. Ewingii species. Therefore, also provided are methods of detectingthe presence of EE polynucleotides in a sample.

Analysis of nucleic acid specific to EE can be by PCR techniques orhybridization techniques (see, for example, Molecular Cloning: ALaboratory Manual, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989; Eremeeva et al., J. Clin.Microbiol. 32:803-810 (1994) which describes differentation amongspotted fever group Rickettsiae species by analysis of restrictionfragment length polymorphism of PCR-amplified DNA). For example, methodsof using nucleic acid probes to analyze EE genomic DNA via PCR analysishave been described in Chen et al., J. Clin. Microbiol. 32:589-595(1994).

In one embodiment, the method includes: a) contacting the sample withthe above-described nucleic acid probe, under speicifc hybridizationconditions such that hybridization occurs and b) detecting the presenceof the probe bound to the nucleic acid molecule, wherein detecting thepresence of such binding is indicative of the presence of E. ewingii inthe sample, and therefore, the subject.

The screening and diagnostic methods of the invention that employ probesdo not require that the entire EE protein coding sequence be used forthe probe. Rather, it is only necessary to use a fragment or length ofnucleiic acid that is sufficient to detect the presence of the EEnucleic acid in a DNA preparation from a subject.

Alternatively, in another embodiment, the method of detecting thepresence of EE nucleic acid in a sample may include: a) amplifying thenucleic acid in the sample with one or more of the above-describedprimer sets specific for one or more portions of the EE omp-1 genecluster, SEQ ID NO: 1 using PCR techniques and b) detecting the presenceof the amplified nucleic acid molecules, wherein the presence of a PCRproduct having a sequence or length which corresponds to the sequence orlength of the portion of the EE omp-1 gene which is located between theprimer set is indicative of the presence of E. ewingii in the sample.

The resulting PCR amplification products can be seperated by size by anymethod, such as gel electrophoresis, and detection of an appropriatelysized product indicates E. ewingii infection. One skilled in the artwould select the nucleic acid probe according to techniques known in theart as described above.

Methods for preparing nucleic acid extracts of cells are well known inthe art and can be readily adapted in order to obtain a sample which iscompatible with the method utilized.

Kits

In another embodiment of the present invention, a kit is provided whichcontains all the necessary reagents to carry out the previouslydescribed methods of detection.

The kit can comprise one or more isolated EE polypeptides. For ease ofdetection, the polypeptides may be attached to a substrate such as acolumn, plastic dish, matrix, or membrane, such as nitrocellulose. Thekit may further comprise a conjugate comprising a binding partner of thepolypeptide. The binding partner can be a biomolecule, such as asecondary antibody, for detecting interactions between the isolatedpolypeptide and antibodies immuno-specific to E. ewingii, in a testsample. In some embodiments, the biomolecule is coupled to a detectabletag such as an enzyme, chromophore, fluorophore, or radio-isotope. Thekit can be used by contacting a test sample with the EE polypeptideunder conditions that permit formation of antigen-antibody complexes.Then the biomolecule is added and the presence or absence of anyresulting antigen-antibody complexes is detected by assaying for achange in the sample, for example, by observing the formation of aprecipitate in the sample, the presence of radioactivity on thesubstrate, or a color change in the sample or on the substrate.Detecting such a change is indicative that the test sample containsanti-E. ewingii antibodies.

In other embodiments the kit can comprise one or more of anabove-described antibodies. The kit can further comprise a conjugatecomprising a binding partner of the antibody. The binding partner can bea biomolecule, such as a secondary antibody, for detecting interactionsbetween the antibodies and the EE OMP protein or peptide in the testsample. In some embodiments, the biomolecule is coupled to a detectabletag such as an enzyme, chromophore, fluorophore, or radio-isotope. Thekit can be used by contacting a test sample with the EE antibody underconditions that permit formation of antigen-antibody complexes. Then thebiomolecule is added and the presence or absence of any resultingantigen-antibody complexes is detected by assaying for a change in thesample, for example, by observing the formation of a precipitate in thesample, the presence of radioactivity on the substrate, or a colorchange in the sample or on the substrate. Detecting such a change isindicative that the test sample contains E. ewingiior components of E.ewingii.

In other embodiments, the above described kits may further comprises oneor more other reagents such as: wash reagents and reagents capable ofdetecting the presence of bound antibodies. Examples of detectionreagents include, but are not limited to, labeled secondary antibodies,or in the alternative, if the primary antibody is labeled, thechromophoric, enzymatic, or antibody binding reagents which are capableof reacting with the labeled antibody.

Also provided are kits for detecting the presence of EE nucleic acid ina sample, which include at least one of the above-described omp-1specific nucleic acid probes or primers. In one embodiment, the kitfurther include: reagents for DNA extraction from the test sample,reagents for PCR amplification, wash reagents and reagents capable ofdetecting the presence of bound nucleic acid probe. Examples ofdetection reagents include, but are not limited to radiolabelled probes,enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase),and affinity labeled probes (biotin, avidin, or steptavidin).

In detail, the kit may be a compartmentalized kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer or reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which container wash reagents (such as phosphate bufferedsaline, Tris-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like.

One skilled in the art will readily recognize that the nucleic acidprobes described in the present invention can readily be incorporatedinto one of the established kit formats which are well known in the art.One skilled in the art will readily recognize that the EE polypeptidesand antibodies described in the present invention can readily beincorporated into one of the established kit formats which are wellknown in the art.

Immunogenic Compositions and Vaccines

The present invention also relates to immunogenic compositionscomprising one or more E. ewingii OMP proteins, or immunogenic fragmentsand variants thereof, or a fusion protein containing same, collectivelyreferred to herein as an “immunogenic EE polypeptide” and apharmaceutically acceptable carrier.

The immunogenic EE polypeptides, as used herein, comprise anepitope-bearing portion of an EE OMP protein. In some embodiments, theepitope-bearing portion comprises a sequence of at least 6 consecutiveamino acids within the variable loops of OMP proteins shown in Tables2-5. Some examples of immunogenic fragments (or peptides) are shown inTable 6.

An immunogenic EE polypeptide is a polypeptide that is capable ofproducing antibodies with a specific binding affinity to E. ewingii in asubject to whom the immunogenic composition has been administered.

In another embodiment, the present invention relates to a vaccinecomprising an immunogenic EE polypeptide, together with apharmaceutically acceptable diluent, carrier, or excipient, wherein theimmunogenic EE polypeptide is present in an amount effective to elicit abeneficial immune response in a subject to EE. The immunogenic EEpolypeptide may be obtained as described above and using methods wellknown in the art.

In another embodiment, the present invention relates to a vaccinecomprising an EE nucleic acid (e.g., DNA) or a segment thereof (e.g., asegment encoding an immunogenic EE polypeptide) together with apharmaceutically acceptable diluent, carrier, or excipient, wherein thenucleic acid is present in an amount effective to elicit, in a subject,a beneficial immune response to EE. The EE nucleic acid may be obtainedas described above and using methods well known in the art.

In a further embodiment, the present invention relates to a method ofproducing an immune response which recognizes EE in a host, comprisingadministering to the host one or more of the above-described immunogenicEE polypeptides.

In some embodiments, the host or subject to be protected is selectedfrom the group consisting of humans, horses, deer, cattle, pigs, sheep,dogs, cats and chickens. In some embodiments, the animal is a human or adog.

In a further embodiment, the present invention relates to a method ofpreventing or inhibiting chrlichiosis in a subject comprisingadministering to the subject the above-described vaccine, wherein thevaccine is administered in an amount effective to prevent or inhibitEhrlichiosis. The vaccine of the invention is used in an amounteffective depending on the route of administration. Althoughintra-nasal, subcutaneous or intramuscular routes of administration aresuitable, the vaccine of the present invention can also be administeredby an oral, intraperitoneal or intravenous route. One skilled in the artwill appreciate that the amounts to be administered for any particulartreatment protocol can be readily determined without undueexperimentation. Suitable amounts are within the range of 2 μg of the EEvaccine per kg body weight to 100 micrograms per kg body weight(preferably, 2 μg to 50 μg, 2 μg to 25 μg, 5 μg to 50 μg, or 5 μg to 10μg).

Examples of vaccine formulations including antigen amounts, route ofadministration and addition of adjuvants can be found in Kensil,Therapeutic Drug Carrier Systems 13:1-55 (1996), Livingston et al.,Vaccine 12:1275 (1994), and Powell et al., AIDS RES, Human Retroviruses10:5105 (1994). The vaccine of the present invention may be employed insuch forms as capsules, liquid solutions, suspensions or elixirs fororal administration, or sterile liquid forms such as solutions orsuspensions. Any inert carrier may be used, such as saline,phosphate-buffered saline, or any such carrier in which the vaccine hassuitable solubility properties. The vaccines may be in the form ofsingle dose preparations or in multi-dose flasks which can be used formass vaccination programs. Reference is made to Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.)(1980); and New Trends and Developments in Vaccines, Voller et al(eds.), University Park Press, Baltimore, Md. (1978), for methods ofpreparing and using vaccines.

The vaccines of the present invention may further comprise adjuvantswhich enhance production of antibodies and immune cells. Such adjuvantsinclude, but are not limited to, various oil formulations such asFreund's complete adjuvant (CFA), the dipeptide known as MDP, saponins(ex. QS-21, U.S. Pat. No. 5,047,540), aluminum hydroxide, or lymphaticcytokines. Freund's adjuvant is an emulsion of mineral oil and waterwhich is mixed with the immunogenic substance. Although Freund'sadjuvant is powerful, it is usually not administered to humans. Instead,the adjuvant alum (aluminum hydroxide) may be used for administration toa human. Vaccine may be absorbed onto the aluminum hydroxide from whichit is slowly released after injection. The vaccine may also beencapsulated within liposomes according to Fullerton, U.S. Pat. No.4,235,877.

The present invention will be better understood by reference to thefollowing examples which are offered by way of illustration, notlimitation.

EXAMPLE—Identification of 19 Polymorphic Major Outer Membrane ProteinGenes and Their Immunogenic Peptides in Ehrlichia Ewingii

Since ehrlichial infections induce significant antibody titers innon-immunocompromised patients, and nonexposed people seldom haveantibodies reactive to Ehrlichia spp., serologic tests are consideredreliable tests for confirmation of ehrlichioses, especially when rulingout the possibility of infection. In order to develop a serologic testusing major antigens of E. ewingii, genes encoding these proteins mustbe first identified.

There are a number of challenges to sequencing genes encoding E. ewingiiouter membrane proteins. First, E. ewingii DNA amount available fromnaturally or experimentally infected dogs is limited. Second, E. ewingiiDNA concentration in the total DNA extracted from the blood is very low,due to a small amount of bacteria present in the blood and is difficultto enrich due to obligatory intracellular nature of this bacterium.Third, DNA sequences encoding OMP-1/P28/P30/MAP1, are too divergent todesign universal primers. Prior to the instant application, only apartial sequence (505 bp) of a single member OMP-1 family p28-19 hasbeen cloned in E. ewingi, and the sequence of other E. ewingii outermembrane proteins, or the genes encoding such proteins, remainedunknown.

The purposes of the reported study were to i) determine the E. ewingiiomp-1 gene family, ii) determine each OMP-1-specific peptide, and iii)analyze all OMP-1 synthesized peptides for antigenicity.

We systematically identified the entire E. ewingii OMP-1 genomic locus.Using nested touchdown PCR and a primer walking strategy, we found 19omp-1 paralogs in E. ewingii. These genes are arranged in tandemdownstream of trl and upstream of secA in a 24-kb genomic region.Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from25.1 to 31.3 kDa with isoelectric points of 5.03 to 9.80.

Those multigene family proteins are composed of conserved and uniqueamino acid sequences. This led to our idea that, rather than the wholeOMP-1 protein, antigenic OMP-1 peptides unique to E. ewingii can providebetter serologic diagnostic antigens. Therefore, differences of thegenomic loci and sequences of E. ewingii omp-1s with those of E.chaffeensis omp-1/p28, E. canis p30s and E. ruminantium map 1 weredetermined to design antigenic OMP-1 peptides specific to E. ewingii.

Based on comparative sequence analyses among OMP-1s from E. ewingii andthe three other Ehrlichia spp. (FIG. 8), each E. ewingii OMP-1oligopeptide predicted to be antigenic, bacterial surface exposed,unique in comparison to the other E. ewingii OMP-1s, and distinct fromother Ehrlichia spp. was synthesized to perform ELISA. Plasma from E.ewingii-experimentally infected dogs significantly reacted with most ofthe OMP-1 specific peptides, indicating that multiple OMP-1 proteinswere expressed and immunogenic in infected dogs. The results support theutility of the tailored OMP-1 peptides as E. ewingii serologic testantigens.

MATERIALS AND METHODS

E. ewingii omp-1 cluster amplification, sequencing, and assembly. AnEDTA-treated whole-blood specimen (−200 pl) collected in April 2005 froman 8-week-old male German Sheperd mixed breed dog in Ohio was used forDNA extraction. DNA was extracted using the QIAamp blood kit (QIAGEN,Valencia, Calif.) and used as the template for the entire amplificationand sequencing process. E. ewingii infection of the dog was confirmed byPCR and sequencing of the 16S rRNA of E. ewingii as well as observationof bacterial inclusions (morulae) in granulocytes in the blood and jointfluid smear. PCR analysis showed that the dog was negative for infectionby A. phagocytophilum, E. chaffeensis and E. canis (Qingming Xiong,Weichao Bao, and Yasuko Rikihisa, unpublished data).

The omp-1 fragments were amplified using first touchdown PCR (Roux, K.H. et al. (1997) Methods Mol. Biol. 67:39-45) with the primer pairs F1and R7, F8 and R14, and F15 and R21 (Table 7). The PCR reaction (50 μl)included 0.5 μl template DNA corresponding to 4 μl of the original bloodsample, 10 pmol of each primer, 0.2 mM deoxynucleoside triphosphatemixture, 2.5 U high-fidelity Taq polymerase (Invitrogen, Carlsbad,Calif.), and 1.5 mM MgC12. Amplification was performed with a program(94° C. for 3 min; a gradient over 10 cycles of 94° C. for 0.5 min, 64°C. for 0.5 min, and 72° C. for 2 min, the annealing temperaturedecreased by 1° C./cycle; 35 cycles of 94° C. for 0.5 min, 55° C. for0.5 min, and 68° C. for 9 min; and finally 68° C. for 9 min). The nestedPCRs were performed using the first PCR products as template with 21pairs of degenerate primers, with amplicons of approximately 1,500 bpthat each overlapped approximately 200 bp according to E. chaffeensisand E. canis omp-1 clusters (Table 7, FIG. 1). Conditions of the nestedPCR were similar to the first PCR except that Taq polymerase was usedand the elongation step was at 72° C. for 2 min. The nested PCR productswere run on a 1% agarose gel with TAE buffer (40 mM Tris-acetate, 1 mMEDTA, pH 8.0). The amplified DNA fragments were recovered from the gelwith the QIAEX II Gel Extraction kit (QIAGEN) and directly sequencedwith the nested PCR primers (FIG. 1). For fragment 3, two touchdown PCRswith high-fidelity Taq polymerase were performed using the infected dogblood DNA as template and one of the following primer pairs: forwardprimer P28-19F and primer R21, or the forward primer designed based onthe 3′ end of fragment 2 (Specific 4F) and reverse primer P28-19R (Table7). Nested amplification of these two PCR products and direct sequencingwere used for subsequent design of new specific primers. Directsequences obtained ranged from 250 to 800 bp. The poly G/C or A/Tregions (FIG. 1) were cloned using the TA cloning kit (Invitrogen), andthe plasmid inserts were sequenced. All sequencing data were assembledusing the SeqMan program of DNASTAR software (DNASTAR Inc., Madison,Wis.)

TABLE 7 Primers used. SEQ SEQ ID ID NO: Degenerate primers NO:E. ewingii specific primers 44 F1: CGYATYATGAGAGGTATGAG  86 Specific 1F:GTACTTTGCCATTCCCAGAGA 45 R1: AGGRTCTATATGTTTTGGTGCT  87 Specific 1R:GATCTACTCCAAACCCAAGAC 46 F2: TTGYATTGGTATAGGGCAAGGA  88 Specific 1RA:GGAATTACTGCTCCAATAGTAGC 47 R2: CTCAAATTTTTTACCRAATAAACCATG  89 Specific2F: GTTGATGGGTATTACCACAGAG 48 F3: CRTATTCATGTTTAGGRTTTGG  90 Specific2R: CACCTAGTATTTTGCTGAAGCT 49 R3: AGTTGCTAWAGCAAARTACTC  91 Specific 3F:TTACTTACCCACTATCTGGTAAC 50 F4: TAGAASTTGAAGCTTTTTATGAG  92 Specific 3R:TAATTTCCCCTGACCTGCAAAC 51 R4: GATATACCRTTRTTTTTTGCTACAG  93 Specific 0F:CAAACCAGTTTATTGACTGGGCAT 52 F5: AARTWCTTTGCTATACCACGTA  94 Specific 19F:CAATCATGCTAAATGCATGTTATGAC 53 R5: TCTATTTCTAYYCTTGGYCCTTG  95 P28 19R:GGATTTATGCTATTAAACATTGTACAC 54 F6: ATRGGYCTTRCAAMTGATGTTAC  96 Specific0FA: TTCAAGCTAAGCTAGGTTTAGG 55 R6: YTTAYTCCARCTTCACCACCA  97 Specific1RB: CATATTAACTCAATCAAGTAAACACAC 56 F7: GCARTAGCWACACTTAATGTTG  98Specific 1FA: CCTCTTACCTCAAATTTAGTTCTC 57 R7: CCTGGTTTATATTGMCCACTT  99Specific 2RA: TTCACCTATACCTAAGCATACATAAG 58 F8: GAGTATTTYGGTRGTGAATTTGG100 Specific 3FA: GTCATGCTATATAGATGATACTGTG 59 R8: RAAATCTCCTCCTAKTCCTGC101 Specific 4F: TCCCTTATGTTTTTGTATTCCTATAC 60 F9:CTGTMATGAGAAAYGACGGGTT 102 Specific 4R: CCATCCATAGCATAACCGATAC 61 R9:TAYYAATKTCAACAGAATCAAYATC 103 Specific 2FA: CTGTTATGAGAAATGACGGAGTTTC 62F10: CAATAYAAACCCAGTGTTTCTG 104 Specific 3FB: CGTACATAGAGTGTTATAGGCAATTC63 R10: GRATAAGTAAYACCTAAYTTACC 105 Specific 0FB:GGTTTAAGTATATGAGTTATAAGAAGGT 64 F11: TAYRGTMAATGGCTGCTATGAT 106 Specific1FB: ATGCACAGGCATTGGTGGAGA 65 R11: AAGTGTAGCWACTGCRGATGT 107 Specific2RB: GTATATATGCATATGTAACATGCAAG 66 F12: TACCATMAAGTAATRGGCAATCA 108Specific 19RA: GGCATGTACTTTCCGCTGATG 67 R12: AYTTCTCCGCCAAAGTATCCA 109Specific 3RA: CTTTACTACTTTCTGATTCACGTAC 68 F13: GCTCCTCAAACCACATCTGC 110Specific 5F: TGCTTTTATTGGTGGGCACTTTC 69 R13: TAKGGTTTATAGCKTCAAACATG 111Specific 6R: TAAGTTTTTTGCATTATCTCGTGAAG 70 F14: TTYTCWCCTTACATATGTGCAG112 Specific 7F: TTGCACAAAAAATCTTTGGCTCAG 71 R14:CARTTCATATTTACACCWGAAAKAGTGAA 113 Specific 7R:ATTAACGCATTTGCATGTAGTAGTGTG 72 F15: GTWTTTAMWTTGTAKKTTTACTACTGTT 114Specific 4FA: CAAGGAAAACTAGGTATAAGTTACTC 73 R15: CTAYCTTGGRCCACCCATTG115 Specific 4RA: AAGACTGGTATGGTAAGACTGTC 74 F16: TAGGGTTTGCAGGAGCTATTG116 Specific 6F: ACACCCCATAACACCACTAAAAG 75 R16:AATTTTAGGRYTTRTAGCTTCAAAC 117 Specific 6RA: GTTTGTTAACTACCCTGTAAAGTC 76F17: TATGYGCAGGTRTTGGTACTGA 118 Specific 7RA: GATAGTACAAACCTGTAAGATGTTAC77 R17: GAWGCTTCTGGGCTTATRGAGT 119 Specific 3RB:AACCTAAATTGCCTATCGATATCATC 78 F18: CAAATCCTAAAATTTCTTAYCAAGGA 120Specific 7RB: TCAACCGTAATATTTAGTGTAGCATC 79 R18:TYAGTAATTTTTCAGCTGAAGAAAC 121 Specific 6FB: CAATATGGCTTTTAGTATCTTGTACATC80 F19: GCAAAAYTGCTTGCATAWGTAG 122 Specific 7RD:TACACTACTTATTGGTATTGTTGGTAG 81 R19: ATTTYTCAGAAGARTATGTTCCA 123 Specific6RA2: TATGTTGTTTGGAGGTGGTTACTATC 82 F20: GAGTMAAAAAYTTTAAYAATRTCTTCTC124 Specific 6FA: CCTATATCTAAGTTAGCTAATGCCGAAG 83 R20:AAAATATCCATTRTAGCTTACCT 125 Specific 7RC: TTTTTGTTTTTCTGTTTTGTGTAACCTGTG84 F21: ATGWTAAATTYATGYTTAAGTTGCA 126 Specific 2FB:CTGGGCATTCTTCAATAGATGCTC 85 R21: SCCYGTYTTCATTTCGGATATC

E. ewingii omp-1 cluster analysis. Artemis (38) was used to identify theORFs in the newly obtained E. ewingii omp-1 cluster. The ORFs longerthan 100 amino acids were blasted against the NCBI GenBank database tofind homologs. The Artemis comparison tool (Carver, T. J., et al. (2005)N Engl J Med 341:148-155) was used to analyze the synteny of the E.ewingii omp-1 cluster to that of E. chaffeensis, E. canis, and E.ruminantium. To search for repeat regions in the E. ewingii omp-1cluster and between the E. ewingii omp-1 cluster and E. chaffeensis, E.canis, or E. ruminantium's omp-1 clusters, dot plot analysis wasperformed with Java Dot Plot Alignments program (JDotter)http://athena.bioc.uvie.ca/index.php.

Phylogenetic analysis. The deduced amino acid sequences of E.chaffeensis OMP1/P28s, E. canis P30s, E. ruminantium MAP1s, and E.ewingii OMP-1s were aligned using the MegAlign program of DNASTARsoftware. Then, phylogenetic analysis was performed with PHYLIP software(version 3.66) (Felsenstein, J. (1989) Cladistics 5:164-166). Thephylogram was constructed using the Neighbor-Joining method withKimura's formula and 1,000 bootstrap replications were conducted toevaluate the reliability of the tree (Felsenstein, J. (1989) Cladistics5:164-166).

Peptide synthesis and peptide-pin ELISA analysis. The peptide librarieswere synthesized using non-cleavable multipin synthesis technology andfluorentylmethoxycarbonyl chemistry (Mimotopes Pty. Ltd., Victoria,Australia) (Geysen, H. M. (1990) Southeast Asian J Trop Med PublicHealth 12:523-533). After disruption of the peptide-pins with 0.1 Msodium phosphate buffer containing 1% SDS (pH 7.2) and 0.1%β-mercaptoethanol and hot (temperature) water washes, nonspecificbinding sites were blocked with 200 μl of 3% skim milk (Becton,Dickinson and Co., Sparks, Md.) in PBS/Tween-20. Blocking was carriedout in 96-well plates for 1 h at room temperature. Sets of peptide-boundpins were washed once with PBS containing 0.1% (v/v) Tween-20 for 10 minand then incubated in the blocking solution (1:100 dilution) with plasmafrom E. ewingii- or E. chaffeensis-infected dogs, or pre-infection dogsplasma, at 4° C. overnight. Samples from dogs 2119, 2185, and 2405 werecollected at days 206, 109, and 110 post-infection, respectively, withE. ewingii. Samples from dog CTUALJ (E. chaffeenis IFA titer, 1:2,560),dog 1425 (E. chaffeensis IFA titer, 1:320), and dog 3918815 (E.chaffeenis IFA titer, 1:2,560) were collected at days 41, 121, andapproximately 210 post-infection, respectively, with E. chaffeensis.Dogs 2119, 2185, and 1425, and preinfection plasma from dog CTUALJ, wereused as negative controls. After washing four times as described above,the peptide pins were placed in wells filled with horseradishperoxidase-labeled goat anti-dog IgG (H+L) (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) diluted at 1:1,000 in PBS/Tween-20 andincubated for 1 h at room temperature. Samples were washed four times,and then the peptide pins were incubated for 20 min at room temperaturewith horseradish peroxidase substrate2,2′-azido-di-(3-ethyl)-benzthiazoline-6-sulfonic acid (Sigma, St.Louis, Mo.) in 70 mM citrate buffer (pH 4.2) applied to a new plate.Absorbance at 405 and 492 nm was measured in an ELISA plate reader(Molecular Devices, Sunnyvale, Calif.). Each assay was repeated at leastthree times. The cut off OD_(405nm)-OD_(492nm) value for positivereaction was set as the mean OD_(405nm)-OD_(492nm)+three standarddeviations of the negative control plasma.

RESULTS

E. ewingii omp-1 cluster sequencing and assembly. Forty-two degenerateprimers were initially designed based on the conserved regions of thealigned omp-1/p30 clusters of E. chaffeensis and E. canis (Table 7, FIG.1). To efficiently utilize the limited amount of E. ewingii DNA, theputative omp-1 cluster was divided into three overlapping fragments ofapproximately 9 kb, estimated based on homologous regions of E.chaffeensis and E. canis. The first touchdown PCR (Roux, K. H. et al(1997) Methods Mol. Biol. 67:39-45) was designed to amplify the threeputative long fragments. The PCR products were then used as templatesfor the 21 nested touchdown PCRs using degenerate primer pairs (Table 7,FIG. 1). As a result only four PCRs showed bands ranging from ˜200 to˜1,500 bp: F1 and R1 (˜700 bp), F4 and R4 (˜1,000 bp), F7 and R7 (˜1,500bp), and F11 and R11 (˜220 bp). The PCR products were directlysequenced. The result showed they belong to the omp-1/p28/p30 family.For regions covered by fragments 1 and 2 (FIG. 1), E. ewingii-specificomp-1 primers were designed based on the four newly obtained E. ewingiiomp-1 DNA sequences (Table 7). However, because no omp-1 was amplifiedin fragment 3 using degenerate primers, two touchdown PCRs withhigh-fidelity Taq polymerase were performed using the infected dog bloodDNA as template and one of the following primer pairs: forward primerP28-19F designed based on the conserved region of E. ewingii p28-19 DNAsequences (Gusa, A. A., et al. (2001) J Clin Microbiol 39:3871-3876) andprimer R21, or the forward primer designed based on the 3′ end offragment 2 and reverse primer P28-19R designed based on the conservedregion on the p28-19 DNA sequence. Nested amplification of these two PCRproducts and direct sequencing were used for subsequent design of newspecific primers. This process was repeated for three fragments until weencountered the poly G/C or A/T regions in fragments 2 and 3. The polyA/T and poly C/G tracts (FIG. 1) were determined by TA cloning andsequencing 10 and 22 plasmid inserts, respectively, in each of tworegions. The poly G tract and 9-13 Gs (the number of Gs was distributedamong the 22 sequenced clones as follows: 9 G=1, 10 G=4, 11 G=7, 12 G=2,and 13 G=8), and was reported as 13 G according to SeqMan software. Thepoly A tract has 10-12 As (the number of As was distributed among the 10sequenced clones as follows: 10 A=1, 11 A=3, 12 A=3, and 13 A=3), andwas reported as 12 A according to SeqMan software. The predominantin-frame sequences in each region were deposited in GenBank. The finalsequence assembled from the entire E. ewingii omp-1 locus contained24,126 bp (GenBank accession No. EF116932). The G+C content of the E.ewingii omp-1 cluster was 28.74%, which is similar to that of E. canis,E. chaffeensis, and E. ruminantium (29.36%, 30.95%, and 27.19%,respectively). E. ruminantium is the causative agent of heartwater inruminants in Africa and Caribbean countries. Sequence identity of theentire E. ewingii omp-1 cluster relative to E. canis, E. chaffeensis,and E. ruminantium was 28.4%, 22.2%, and 14.8%, respectively.

Features of the OMP Cluster Structure are Conserved Among the EhrlichiaSpecies

The Artemis software analysis showed that each of the 24 ORFs encodemore than 100 amino acids in the assembled E. ewingii omp-1 DNAfragment. One of the 24 ORFs in the middle of the cluster was short (390bp), partially overlapped with two other ORFs in the oppositeorientation, and had no homolog in the GenBank database, and thus thisORF was not included in the Figures or Table 1. The 23 ORFs were numbersORF 1 to 23. These 23 genes were arranged in tandem except for threeORFs (ORF19, 20, and 21) that were in the opposite orientation. Nineteenof these 23 ORFs encoded proteins homologous to OMP-11P28/MAP1 of E.chaffeensis, E. canis, or E. ruminantium. Most closely related proteinsto each EEOMP-1 are listed in Table 8.

We numbered them E. ewingii (EE)OMP-1-1 to EEOMP-1-19 (FIG. 2). Thesequence similarity and molecular mass of EEOMP-1-8 was less than thatof the other EEOMP-1s. There is a protein ortholog of EEOMP-1-8, UN3, inthe E. chaffeensis and E. canis genomes with unknown function. In E.ruminantium, the EEOMP-1-8 ortholog is MAP1-9. The protein encoded bythe first ORF (ORF1) is homologous to a hypothetical transcriptionalregulator, and the protein encoded by the last ORF (ORF23) is homologousto SecA. Proteins encoded by the other two ORFs (ORF4 and ORF22) aremost homologous to two E. chaffeensis and E. canis peptides, UN2 andUN4, with unknown function, as well as two E. ruminantium peptides, UN1and UN2, whose function is unknown. The p28-19 505 bp sequence was apart of EEomp-1-16 (Table 1). Intergenic spaces between omp-1 genesranged from 6 to 1,343 bp (Table 1). At the 5′ half of each OMP cluster,14 genes (un2 to EEomp-1-13 in E. ewingii) were linked by shortintergenic spaces ranging from 6 to 26 bp (Table 1). Eight genes in the3′ half (EEomp-1-14 to EEomp-1-19) were connected by longer intergenicspaces ranging from 301 to 808 bp. Thus, features of the OMP clusterstructure were conserved among E. ewingii, E. canis, E. chaffeensis, andE. ruminantium, with the exception of the opposite orientation of threegenes, instead of one gene (E. canis and E. ruminantium) or two genes(E. chaffeensis) at the 3′end.

After removal of the signal peptide sequence, predicted molecular massesof mature E. ewingii OMP-is ranged from 25.1 to 31.3 kDa. The predictedsignal peptides ranged from 21 to 32 amino acids. The predictedisoelectric points of the mature OMP-is were 5.03 to 9.80. Properties ofthe ORFs of the E. ewingii omp-1 cluster, including predicted signalpeptide lengths, molecular masses of mature proteins and isoelectricpoints, are shown in Table 1.

omp-1/p28/map1 gene clusters display synteny at the 5′ end. The syntenyamong entire OMP-1 gene clusters of E. ewingii and three relatedEhrlichia species was analyzed by Artemis Comparison Tool, and theresults are shown in FIG. 3. The genes at the 5′ end of the omp-1clusters were more highly conserved than genes in the central region or3′ end (FIG. 3). Previously we defined three repeat sequence regions, α,βand γ, in omp-1 clusters of E. chaffeensis and E. canis (Ohashi, N., etal. (2001) Infect Immun 69:2083-2091). The dot plot analysis of the E.ewingii omp-1 cluster and the dot plot between E. ewingii and E.ruminantium only revealed β and γ repeat regions (FIG. 4). The β repeatregion in E. ruminantium was shorter than that of E. chaffeensis and E.canis. The dot plot analysis between E. ewingii and E. chaffeensis andbetween E. ewingii and E. canis showed three clear repeat regions,indicating that the a region is expanded in E. canis and E. chaffeensis.

The phylogenetic analysis of all 79 OMPs of E. ewingii, E. chaffeensis,E. canis, and E. ruminantium is shown in FIG. 5. The previously defineda and β1 regions in the E. chaffeensis omp-1 cluster (Ohashi, N., et al.(2001) Infect Immun 69:2083-2091) encoded five (P28, OMP-1F, -1D, -1C,and -1E) and four (OMP-1H, -1A, -1S, and -1Z) proteins, respectively,and α and β1 regions in the E. canis p30 cluster encoded six (P30,P30-1, P30-2, P30-3, P30-4, and P30a and four (P30-6, P30-5, P30-7, andP30-8) proteins, respectively. However, in E. ewingii, the α and β1regions each encoded two proteins (EEOMP-1-15, EEOMP-1-16 andEEOMP-1-12, EEOMP-1-13, respectively). In E. ruminantium, the α regionencoded only one protein (MAP1) and the β1 region encoded two proteins(MAP1-2, MAP1-3) (FIG. 5)

EEOMP-1-8 and MAP1-5 were far removed from the remaining OMP-1s, raisingthe possibility that they do not belong to the OMP cluster (FIG. 5).EEOMP1-18 and EEOMP-1-19, which are encoded by genes in the reverseorientation, were clustered with P28-2, which is encoded by one of tworeverse-oriented E. chaffeensis omp-1 genes. All proteins except α andβ1 group proteins formed separate small clusters, including fourproteins from each of the four Ehrlichia species. Each cluster ofproteins is thus expected to share a common ancestor.

Previously reported 505-bp E. ewingii p28-1 sequences (GenBank accessionnumbers: AF287961, AF287962, AF287963, AF287964, AF287966) (Gusa, A. A.,et al. (2001) J Clin Microbiol 39:3871-3876) were compared withcorresponding sequences identified in the present study. The 505 bpbegins at 16,918 bp and ends at 17,422 bp in the cluster, whichcorresponds to 75% of omp-1-16 (i.e., from 123 to 637 bp of the 849-bpomp-1-16 gene). The E. ewingii p28-1 sequences of a Missouri caninesample and an Oklahoma human sample (Gusa, A. A., et al. (2001) J ClinMircobiol 39:3871-3876) were identical to the sequence obtained from theOhio dog analyzed in the present study.

TABLE 8 Comparison of the most closely related E. chaffeensis and E.canis OMPs with E. ewingii OMPs E. ewingii OMP-1 Most closely related %identity of paralogs Ehrlichia orthologs orthologs OMP-1-1 OMP-1M 66.2OMP-1-2 OMP-1N 51.5 OMP-13 OMP-1Q 45.1 OMP-1-4 OMP-1P 51.6 OMP-1-5OMP-1T 48.5 OMP-1-6 P30-14 60.9 OMP-1-7 OMP-1V 67.4 OMP-1-8 MAP1-8 18.9OMP-1-9 P30-12 51.6 OMP-1-10 P30-11 59.5 OMP-1-11 OMP-1Y 55.8 OMP-1-12P30-5 63.5 OMP-1-13 OMP-1H 59.4 OMP-1-14 OMP-1B 71.0 OMP-1-15 OMP-1E60.8 OMP-1-16 OMP-1F 64.3 OMP-1-17 P28-1 69.4 OMP-1-18 P28-2 49.3OMP-1-19 P28-2 47.1

E. ewingii OMP-1-specific peptide ELISA. Following our OMP-1 amino acidsequence alignment, repetitive sequence analysis, and phylogenicanalysis results, we designed E. ewingii OMP-1-specific peptides forserologic tests. As OMP-1s share repetitive common or homologous aminoacid sequences with OMP-1s of the same or different Ehrlichia sp., it isdifficult to design recombinant proteins (>10 kDa) that provideEhrlichia sp.-specific or gene-specific antigens. Also, to clone,express, and purify 19 recombinant OMP-1 proteins are cost andlabor-prohibitive. Therefore, we designed 12-17-mer peptides specific toeach of the 19 E. ewingii OMP-is. For this purpose, extracellular loopsof the 19 E. ewingii OMP-1s were first predicted using the PosteriorDecoding method of PRED-TMBB(http://bioinformatics.biol.uoa.gr/PRED-TMBB) (Bagos, P. G., et al.(2004) Nucleic Acids Res 32:W400-404). PRED-TMBB is a web server capableof predicting transmembrane strands and topology of β-barrel in OMPs ofGram-negative bacteria based on a Hidden Markov Model. The validity ofthese predictions is tested using non-homologous OMPs with structuresknown at atomic resolution according to the Conditional MaximumLikelihood criteria (Bagos, P. G., et al. (2004) Nucleic Acids Res32:W400-404). Relatively highly antigenic and hydrophilic 12-17-merpeptide fragments located within one of the extracellular loops werechosen from each of the 19 EEOMP-1 amino acid sequences based on DNASTARProtean analysis. Using the program BLAST, these peptide sequences werecompared with the entire E. ewingii omp-1 locus and the E. chaffeensis,E. canis and E. ruminantium genoma sequences to synthesize one peptidespecific to each of the 19 EEOMP-1 (FIG. 8, Table 6).

Plasma from three dogs experimentally infected with E. ewingii andpreinfection plasma from four dogs were then tested in ELISA containingthe 19 EEOMP-1-specific peptides. Thirteen peptides (EEOMP-1-1,EEOMP-1-2, EEOMP-1-3, EEOMP-1-4, EEOMP-1-5, EEOMP-1-8, EEOMP-1-9,EEOMP-1-10, EEOMP-1-13, EEOMP-1-14, EEOMP-1-15, EEOMP-1-16, andEEOMP-1-19) were consistently recognized with plasma from three dogsexperimentally infected with E. ewingii compared with preinfection dogplasma (FIG. 6).

As geographical distributions, vector ticks and animal reservoirsoverlap between E. ewingii and E. chaffeensis, it is important todistinguish them by a simple assay. Therefore, we examined theimmunological cross reactivity of these peptides with plasma from threedogs experimentally infected with E. chaffeensis. Among 13 EEOMP-1sspecifically recognized in E. ewingii-infected dogs, EEOMP-1-8,EEOMP-1-10, and EEOMP-1-15 were recognized by one of three E.chaffeensis-infected dogs (FIG. 6). While more specimens need to betested, the peptide-pin ELISA result suggests that of the remaining 10EEOMP-1 peptides, 8 peptides (EEOMP-1-1, EEOMP-1-3, EEOMP-1-4,EEOMP-1-5, EEOMP-1-13, EEOMP-1-14, EEOMP-1-16, and EEOMP-1-19) serve asgood candidate antigens for E. ewingii serodiagnosis based on highsensitivity (indicated by the ratio of E. ewingii plasmareactivity/control plasma reactivity) and good specificity (indicated bythe ratio of E. chaffeensis plasma reactivity/control plasma reactivity,−1.00).

DISCUSSION

In the present study and for the first time, the entire 24-kb E. ewingiiomp-1 locus containing 19 omp-1 genes was sequenced. As the onlyavailable source of E. ewingii DNA was a small amount of the infecteddog blood specimen, we employed touchdown PCR. This method has been usedpreviously to amplify a small amount of fragmented Aegyptianellapullorum DNA from archival paraffin sections on glass slides. Incorrectbase calls resulting from amplification or sequencing errors have beenminimized in the present study because large pools of PCR products weredirectly sequenced. In addition, multiple overlapping regions throughoutthe sequences ensure the reliability of sequencing results. So far, onlya few E. ewingii genes, including 16S rRNA, groESL, p28-19, dsbA(GenBank Accession No. DQ902688), gltA (GenBank Accession No. DQ365879),and disulfide oxidoreductase have been reported. Applying a similarapproach as used here, it would be possible to obtain DNA sequences ofother genomic regions to further our understanding of this uncultivableemerging zoonotic pathogen.

Because E. ewingii infects granulocytes, the distinction between E.ewingii and a strain of A. phagocytophilum was unclear prior to themolecular era. However, in concordance with the 16S rRna and groESLsequence-based classification of this bacterium, our finding of thecomplete OMP-1 cluster structure flanked with trl and secA clearlydemonstrated that E. ewingii belongs to the genus Ehrlichia. Syntenyanalysis suggests that the OMP clusters existed in a common ancestor ofthe present day four Ehrlichia species. Furthermore, the locus appearsto have been partially scrambled as species evolved. The E. ewingiiOMP-1 cluster has greater synteny with monocytotropic E. chaffeensis andE. cards than with the endotheliotropic E. ruminantium. It is possiblethat OMP-1s and host cell type specificity co-evolved.

The present study revealed 19 E. ewingii OMP-1 amino acid sequences andexamples of 19 E. ewingii immunogenic amino acid sequences. Studies onE. chaffeensis have shown an important role for OMP-1/P28 outer membraneproteins in the stimulation of host immune response and protection ofthe host from infection. Immunization with recombinant P28 (one of themajor outer membrane OMP-1/P28 family members) has been shown to protectmice from E. chaffeensis challenge. The monoclonal antibody againstOMP-1 g (P28) mediates protection of SCID mice from E. chaffeensis fatalinfection. While antibodies against a single OMP-1 protein conferpartial protection, existence of multiple homologous surface proteinslikely plays a role in the organism's evasion of host immune response. Arecent proteomic study showed that 18 out of 21 E. chaffeensis OMP-1/P28family proteins are indeed bacterial surface-exposed, supporting theidea of immunoevasion. The number of E. ewingii omp-1 genes found in theOMP-1 cluster (19 copies) was similar to that of E. canis (22 copies,but there is an additional locus with duplicates of three p30s) E.chaffeensis (22 copies), and E. ruminantium (16 copies). In addition,there is extensive diversification among omp-1 genes of E. ewingii,similar to other Ehrlichia spp., supporting the hypothesis that multipleomp-1/p28 paralogs present in Ehrlichia wp. are involved inimmunoavoidance. Thus, theses studies suggest that incorporation ofimmunogenic peptides of multiple OMP-1s in the vaccine preparation mayprovide better protection against Ehrlichia infection than the use of asingle OMP-1 in the vaccine.

Multiple OMP-1/P28 and P30 mRNAs are expressed by E. chaffeensis and E.canis during experimental infections of dogs with these bacteria. All 22E. chaffeensis P28 recombinant antigens are recognized by sera from twodogs experimentally infected with E. chaffeensis. Similarly, the presentresults suggest all 19 EEOMP-1 peptides were recognized by the plasmafrom three E. ewingii-infected dogs. Thus, the lack of immunlogicalcross-reactivity of E. canis and E. chaffeensis OMP-1/P28/P30 withplasma from human patients or dogs infected with E. ewingii in theprevious studies is likely due to divergence of the amino acid sequencesof the E. ewingii OMP-is from those of the E. canis and E. chaffeensisOMP-1/P28-P30 proteins expressed in cell culture. It is also most likelythat in E. ewingii infected humans and dogs, different combinations ofmultiple OMP-1s are expressed at different stages of infection, andunder different immune and health status of animals. Therefore, forserodiagnosis of E. ewingii infection in both humans and animals, use ofa combination of EEOMP-1 peptides as serodiagnostic antigens is expectedto provide more sensitive and more specific serodiagnosis with broadercoverage than the use of a single EEOMP-1 antigen. Furthermore, the DNAsequence data also obtained in the present study should help refinediagnostic PCR for human and dog granulocytic ehrlichioses to make thisdirect test more reliable for all infective species.

The invention claimed is:
 1. A method for detecting antibodies specificfor E. ewingii (EE), comprising: (a) contacting a test sample with oneor more isolated EE polypeptides under conditions that allowpolypeptide/antibody complexes to form; and (b) assaying for theformation of a complex between antibodies in the test sample and the oneor more EE polypeptides; wherein the formation of said complex is anindication that antibodies specific for E. ewingii are present in thetest sample; and wherein at least one of the one or more EE polypeptidescomprises an amino acid sequence that comprises: 1) 6 or moreconsecutive amino acids of one or more amino acid sequences of variableregion loops 1-4 of a mature OMP-1-19 protein encoded by nucleotide21188-21967 of SEQ ID NO:1, or 2) 6 or more consecutive amino acids ofthe amino acid sequence set forth in SEQ ID NO: 43, and wherein ananti-E. ewingii antibody has a specific binding affinity for the atleast one of the one or more isolated EE polypeptides.
 2. The method ofclaim 1, wherein the one or more amino acid sequences of the variableregion loops 1-4 comprises 6 or more consecutive amino acids of an aminoacid sequence that is set forth in SEQ ID NO: 155, SEQ ID NO: 173, SEQID NO: 191, SEQ ID NO:208 and/or SEQ ID NO:227.
 3. The method of claim1, wherein the one or more EE polypeptides comprises 6 or moreconsecutive amino acids of the amino acid sequence that is set forth inSEQ ID NO:
 43. 4. The method of claim 1, wherein the one or more EEpolypeptides comprises an amino acid sequence of a mature OMP-1-19protein that comprises amino acids 24 to 282 of SEQ ID NO:
 22. 5. Themethod of claim 1, wherein the one or more EE polypeptides comprises anamino acid sequence of a mature OMP-1-19 protein that consists of aminoacids 24 to 282 of SEQ ID NO:
 22. 6. The method of claim 1, wherein theone or more EE polypeptides further comprises an amino acid sequencethat comprises 6 or more consecutive amino acids of a mature OMP-1-3protein having the amino acid sequence from residue 24-284 of SEQ ID NO:6, a mature OMP-1-10 protein having the amino acid sequence from residue26 to 280 of SEQ ID NO: 13, a mature OMP-1-15 protein having the aminoacid sequence from residue 26 to 278 of SEQ ID NO: 18, a mature OMP-1-16protein having the amino acid sequence from residue 26 to 282 of SEQ IDNO: 19, or any combination thereof.
 7. The method of claim 1, whereinthe one or more EE polypeptides comprises an amino acid sequence thatcomprises 6 or more consecutive amino acids of a mature OMP-1-15 proteinhaving the amino acid sequence from residue 26 to 278 of SEQ ID NO: 18,a mature OMP-1-19 protein having the amino acid sequence from residue 24to 282 of SEQ ID NO: 22, or a combination thereof.
 8. The method ofclaim 1, wherein the one or more EE polypeptides comprises an amino acidsequence that comprises 6 or more consecutive amino acids of a matureOMP-1-10 protein having the amino acid sequence from residue 26 to 280of SEQ ID NO: 13, a mature OMP-1-15 protein having the amino acidsequence from residue 26 to 278 of SEQ ID NO: 18, a mature OMP-1-16protein having the amino acid sequence from residue 26 to 282 of SEQ IDNO: 19, a mature OMP-1-19 protein having the amino acid sequence fromresidue 24 to 282 of SEQ ID NO: 22, or any combination thereof.
 9. Themethod of claim 1, wherein the one or more EE polypeptides isoperatively linked to an N-terminal or C-terminal peptide or tag. 10.The method of claim 1, wherein the one or more EE polypeptides is arecombinant form of the EE polypeptide(s).
 11. The method of claim 1,wherein the isolated EE polypeptide is attached to a substrate.
 12. Themethod of claim 11, wherein the substrate is a column, plastic dish,matrix, or membrane.
 13. The method of claim 12, wherein the membranecomprises nitrocellulose.
 14. The method of claim 1, wherein the testsample is untreated, or subjected to precipitation, fractionation,separation, or purification before combining with the EE polypeptide(s).15. The method of claim 1, wherein the formation of a complex betweenantibodies in the test sample and the one or more EE polypeptides isdetected by radiometric, calorimetric, or fluorometric means, or bysize-separation or precipitation.
 16. The method of claim 1, which isused in an assay format selected from the group consisting of amicrotiter plate assay, a reversible flow chromatographic binding assay,an enzyme linked immunosorbent assay, a radioimmunoassay, ahemagglutination assay, a western blot assay, a fluorescencepolarization immunoassay, an indirect immunofluorescence assay, adiffusion based Ouchterlony, and a rocket immunofluorescent assay. 17.The method of claim 1, wherein the formation of a complex betweenantibodies in the test sample and the one or more EE polypeptides isdetected by addition of a secondary antibody that is coupled to adetectable tag.
 18. The method of claim 17, wherein the detectable tagis an enzyme, a fluorophore, or a chromophore.
 19. The method of claim1, which is used to determine whether a subject is infected with E.ewingii.
 20. The method of claim 1, wherein the subject is a human. 21.The method of claim 1, wherein the subject is an animal.
 22. The methodof claim 21, wherein the animal is a horse, a deer, a cattle, a pig, asheep, a dog, a cat or a chicken.
 23. The method of claim 1, wherein theone or more amino acid sequences of the variable region loops 1-4comprises an amino acid sequence that is set forth in SEQ ID NO: 155,SEQ ID NO: 173, SEQ ID NO: 191, SEQ ID NO:208 and/or SEQ ID NO:227. 24.The method of claim 3, wherein the one or more EE polypeptides comprisesthe amino acid sequence that is set forth in SEQ ID NO:43.
 25. Themethod of claim 6, wherein the one or more EE polypeptides furthercomprises an amino acid sequence that comprises 6 or more consecutiveamino acids of the amino acid sequence set forth in SEQ ID NO: 27, theamino acid sequence set forth in SEQ ID NO: 34, the amino acid sequenceset forth in SEQ ID NO: 39, the amino acid sequence set forth in SEQ IDNO: 40, or any combination thereof.
 26. The method of claim 7, whereinthe one or more EE polypeptides comprises an amino acid sequence thatcomprises 6 or more consecutive amino acids of the amino acid sequenceset forth in SEQ ID NO: 39, the amino acid sequence set forth in SEQ IDNO: 43, or a combination thereof.
 27. The method of claim 8, wherein theone or more EE polypeptides comprises an amino acid sequence thatcomprises 6 or more consecutive amino acids of the amino acid sequenceset forth in SEQ ID NO: 34, the amino acid sequence set forth in SEQ IDNO: 39, the amino acid sequence set forth in SEQ ID NO: 40, the aminoacid sequence set forth in SEQ ID NO: 43, or any combination thereof.